JP2022519067A - Sensor chip and its method - Google Patents

Abstract

The present disclosure generally relates to sensor chips and methods for detecting specimens. Specifically, the present disclosure relates to a sensor chip for detecting a sample in a subject suffering from a neurodegenerative disease. The sensor chip includes a conductive layer on the membrane support layer, and a plurality of apertures penetrate the conductive layer and the membrane support layer, and the plurality of apertures irradiate the conductive layer and / or the membrane support layer. Is arranged so that surface plasmon resonance occurs. TIFF2022519067000007.tif60156

Description

Cross-reference to related applications This application claims the priority of Singapore Patent Application No. 10201900937Q, named SENSOR CHIP AND METHODS THE REOF, filed January 31, 2019.

Field This disclosure generally relates to sensor chips and methods for detecting specimens. Specifically, the present disclosure relates to sensor chips that detect specimens in subjects suffering from neurodegenerative diseases and other diseases such as amyloidosis.

Background Surface plasmon resonance (SPR) detection is a widely used technique in laboratories to characterize interactions between biomolecules, such as antibodies and antigens. This technique is typically based on immobilizing a ligand capture molecule on a metal surface and measuring the change in index of refraction when the ligand binds to the capture molecule. This technique is a labeling-free technique and does not require the use of special tags or dyes for sensitive measurements of intramolecular interactions. Currently, it is being developed for use in laboratories for the diagnosis of patients with various pathologies such as dementia, hepatitis, diabetes, and cancer.

Dementia is a public health crisis in the 21st century. Severe dementia, the most common form of Alzheimer's disease (AD), is characterized by progressive, loss of memory and cognitive function. Affected individuals have significant restrictions on self-care, social and occupational functioning. However, the molecular features of AD may manifest and progress long before these full-blown clinical symptoms appear. They include extracellular amyloid β (Aβ) plaques and intracellular tau neurofibrillary tangles. Due to the complexity and progression of this neuropathology, early detection and intervention are considered essential for successful disease modification treatment.

However, current AD diagnosis and disease observation are subjective and are performed late. They are made by clinical and neuropsychological assessments using published criteria. Such an approach lacks sensitivity and specificity, especially in the early stages when the symptoms are subtle and overlap well with a variety of other disorders. New molecular diagnostic assays are under development, such as cerebrospinal fluid measurement and positron emission tomography (PET) imaging of amyloid plaques in the brain, either because these tests require invasive lumbar puncture or. It faces limitations because it is too expensive for widespread clinical application. As a result, there is a great deal of interest in finding serum biomarkers for AD to aid in early diagnosis and disease observation.

Therefore, one or more of the above problems need to be overcome, or at least alleviated.

This specification discloses a sensor chip and a method for detecting a sample by surface plasmon resonance.

In one aspect, a sensor chip comprising a conductive layer on a membrane support layer is provided in which a plurality of apertures penetrate the conductive layer and the membrane support layer, and the plurality of apertures are the conductive layer and / or the same. It is arranged so that surface plasmon resonance occurs due to irradiation of the membrane support layer.

In one aspect, an imaging system comprising a light source, a detector, and a sensor chip as defined herein is provided, the light source being arranged to illuminate the sensor chip, the detector being the same. It is positioned to detect the light transmitted through the sensor chip.

In one aspect, a method of manufacturing a sensor chip is provided, which method is:
(a) Step of providing the upper membrane support layer;
(b) A step of depositing a conductive layer on the upper membrane support layer;
(c) Including the step of forming a plurality of apertures penetrating the membrane support layer, the plurality of apertures also penetrate the conductive layer and are surfaced by irradiation of the conductive layer and / or the superficial membrane support layer. It is arranged so that plasmon resonance occurs.

In one aspect, a method of detecting a sample in a sample is provided, wherein the method is:
(a) The step of capturing the sample on the surface of the sensor chip as defined herein;
(b) Including the step of detecting the binding between the second recognition molecule and the sample captured on the surface of the sensor chip, the second recognition molecule is specific to the sample and compared with the control sample. The increased binding of the second recognition molecule indicates the presence of the sample in the sample.

In one aspect, a kit comprising the sensor chips as defined herein is provided.

In one aspect, a method of detecting a neurodegenerative disease or amyloidosis of interest is provided, wherein the method is:
(a) The step of bringing the sample taken from the subject into contact with the surface of the sensor chip as defined herein;
(b) The second recognition molecule comprises the step of detecting the binding between the second recognition molecule and the sample captured on the surface of the sensor chip, the second recognition molecule is specific to the sample, and the sample taken from the control target. The increased binding of the second recognition molecule in comparison with the subject indicates that the subject suffers from a neurodegenerative disease.

Also provided is the use of the sensor chips described herein in a method of detecting a neurodegenerative disease or amyloidosis of interest.

Next, some aspects of the invention will be described as merely non-limiting examples with reference to the accompanying drawings.

Figure 1: APEX platform for circulating exosome-bound Aβ analysis. (A) Exosomes bind to Aβ protein. The Aβ protein, the main component of amyloid plaque found in AD brain pathology, is released into the extracellular space. Exosomes are nanoscale outer membrane vesicles that are actively secreted by mammalian cells. Exosomes can bind to released Aβ proteins by their surface glycoproteins and glycolipids. (B) Transmission electron micrograph of exosome-bound Aβ. Exosomes from neurons (SH-SY5Y) were treated with Aβ42 aggregates and labeled with gold nanoparticles (10 nm) via Aβ42 specific antibodies. Nanoparticles are visible as black dots (indicated by red arrows). (C) Outline of APEX assay. To enable nanoscale sensitive profiling, exosomes are first immunocaptured by a plasmon nanosensor (before amplification). Enzymatic amplification of in situ causes insoluble light deposits to localize on sensor-bound exosomes (after amplification). This adhesion is spatially defined for molecular co-localization analysis and alters the index of refraction for SPR signal enhancement. In addition, in order to complement the enzymatic amplification, the nanosensor is back-illuminated (away from the enzymatic activity) to achieve analytical stability. As a result of this adhesion, a redshift of light transmitted through the nanosensor is obtained. (D) Typical outline of transmission spectrum change due to APEX amplification. Specific exosome binding (before) and subsequent amplification profiling (after) were observed as transmission spectrum shifts (Δλ) by the APEX platform. a.u .: Arbitrary unit. (E) Exosome-bound Aβ in blood samples of Alzheimer's disease (AD), mild cognitive impairment (MCI), and non-cognitive (NCI) control controls were measured using the APEX platform. This blood measurement was correlated with PET imaging of the corresponding cerebral amyloid plaque deposits. (F) Photograph of APEX microarray. Each sensor chip contains 6 x 10 detector elements made of uniformly manufactured plasmon grids for complex measurements. See Figures 7-10 for sensor manufacturing, feature determination, and design optimization, respectively. See the description in Figure 1-1. APEX signal amplification and combined profiling. (A) Gradual change in APEX transmission spectrum. A series of operations, antibody (anti-CD63) binding, exosome binding, enzymatic labeling, and enzymatic attachment to the sensor were performed and the resulting spectral shift was observed. Enzyme labeling did not result in significant changes, but deposit formation resulted in approximately 400% signal enhancement (**** P <0.0001, n.s .: no significant difference, Student's t-test). (B) Comparison between APEX signal amplification and the area range of light deposits. The increase in area range was determined by scanning electron microscopy (SEM) analysis (**** P <0.0001, Student's t-test). All data were normalized to those before signal amplification. Inserts (right) show SEM images of sensor-bound exosomes before and after APEX amplification. (C) Finite difference time domain simulation using backside illumination. The APEX sensor design allows the generation of electromagnetic fields enhanced by backside illumination, but the gold-supported glass design does not. Backside illumination minimizes the direct exposure of enzyme activity (generated on the top surface of the sensor) to incident light. Arrows indicate the incident direction of irradiation. (D) Real-time sensorgram of APEX amplification dynamics. Amplification efficiency was observed using optical substrates with different concentrations (3,3'-diaminobenzidine tetrahydrochloride; high: 1 mg / ml, low: 0.01 mg / ml). All data were normalized to negative controls performed with IgG isotope control antibodies. (E) Comparison of detection sensitivities of APEX, ELISA, and Western blotting. The APEX detection limit (dotted line) before and after amplification was determined by titration of known amounts of exosomes and measurement of their CD63 signal. (F) Target protein measurement specificity of APEX assay. Amyloid β (Aβ42), amyloid precursor protein (APP), alpha-synuclein (α-syn), L1 closely related homologue (CHL1), insulin receptor substrate 1 (IRS-1), nerve cell adhesion molecule (NCAM), And developed an assay for tau protein. Specific detections were demonstrated in all assays. The heat map signal normalized the assay (row). All measurements are performed in triplets and the data are shown as mean ± standard deviation for a, b, d, and e. Figure 3: Preferential binding of Aβ aggregates to exosomes. (A) Outline of Aβ protein aggregation. Small Aβ42 aggregates and large Aβ42 aggregates were prepared by varying the degree of clustering and using filtration. (B) Characterization of Aβ protein aggregates. (Left) Transmission electron micrographs show the spherical morphology of the prepared Aβ42 aggregates. (Right) Dynamic light scattering analysis confirmed the monopeak size distribution of preparations of different sizes. (C) Outline of exosome-Aβ binding analysis. Aβ42 aggregates (small vs. large) were immobilized on APEX sensors and treated with equal concentrations of neuronal cell (SH-SY5Y) -derived exosomes to determine binding kinetics. All exosome binding data were normalized to the surface area of each Aβ42 aggregate immobilized on the sensor (see Methods for details). (D) Real-time sensorgram of exosome binding kinetics. Exosomes bound more strongly to Aβ42 aggregates compared to similarly sized bovine serum albumin (BSA) control aggregates (see Figure 13). Importantly, exosomes demonstrated a much stronger affinity for larger Aβ42 aggregates (right) compared to binding affinity for smaller Aβ42 aggregates (left). Note the difference in scale on the y-axis. All binding affinities (KD) were determined from normalized exosome binding data and relative to BSA controls. KD (small) / KD (large) = 5.27. (E) Differences in binding between various extracellular vesicles and Aβ42 aggregates. The vesicles were derived from various cell origins, namely neurons, glial cells, endothelial cells, monocytes, erythrocytes, platelets, and epithelial cells, respectively, and were used for binding analysis at equal concentrations. First, the APEX platform was used to measure the direct binding of each vesicle to the Aβ42 functionalized sensor (directly). Next, for each cell origin, label the bound vesicle origin specific marker (cell origin specific marker) or pan-exosome marker (ie, CD63, pan-exosome marker) and measure the associated APEX signal amplification. did. All measurements were relativized to IgG isotope control antibody and performed in triplets. In e, the data is shown as mean ± standard deviation. See the description in Figure 3-1. See the description in Figure 3-1. Clinical correlation between circulating exosome-binding Aβ and brain imaging. (A) It is a typical restored PET brain image of clinical subjects, and it can be seen that the cerebral amyloid plaque load increases. The standardized uptake value ratio SUVR for specific brain regions was normalized relative to the average cerebellar gray material intensity to determine brain amyloid plaque loading. (B) Correlation between different circulating Aβ42 populations and global mean PET brain imaging (n = 72). Patients with Alzheimer's disease (AD), mild cognitive impairment (MCI), and controls for non-cognitive impairment (NCI), vascular dementia (VaD), and vascular mild cognitive impairment (VMCI) using the APEX assay. Signals from exosome-bound Aβ42 (left), unbound Aβ42 (center), and total Aβ42 (right) in the subject's blood sample were measured, respectively. Correlated with global imaging data of brain amyloid plaques, exosome binding compared to measurements of the unbound Aβ42 population (center, R ² = 0.0193) or total Aβ42 measurements (right, R ² = 0.1471). It was demonstrated that the Aβ42 measurements were the most correlated (left, R ² = 0.9002). (C) Analysis of different circulating Aβ42 populations regarding the distinction between different clinical groups (n = 84). Only APEX measurements of circulating exosome-bound Aβ42 (left) can distinguish between AD clinical groups (AD and MCI) and also from other healthy controls (NCI) and clinical controls (VaD, VMCI, and acute stroke). rice field. Neither unbound Aβ42 measurements (center) nor total Aβ42 measurements (right) showed statistical significance between different clinical groups (** P <0.01, **** P <0.0001, ns). : No significant difference, Student's t-test). All measurements were relativized to IgG isotope control antibody and performed in triplets. In b to c, the data are shown as mean ± standard deviation. Characterizing extracellular vesicles released from nerve cells. (A) Scanning electron micrographs of neurons (SH-SY5Y) showing active release of nanoscale extracellular vesicles from cells. (B) High-magnification image of the released vesicles. (C) Single peak size distribution of extracellular vesicles showing an average diameter of approximately 150 nm as determined by nanoparticle tracking analysis. (D) Western blotting analysis of vesicle lysate. It lyses vesicles and lyses exosome markers (LAMP 1, ALIX, HSP90, HSP70, CD63, flotilin 1, TSG101), neuronal markers (NCAM), and markers of lipoproteins (APOE) and other membrane compartments (carnexin, Immunoblots were performed for negative markers such as GRP94). (E) Transmissive electron micrographs of dual immunolabels (CD63, 20 nm; Aβ42, 5 nm) with different sized gold nanoparticles showing the presence of exosome-bound Aβ, both markers on the same vesicle. Co-localization was confirmed. APEX amplification product. Scanning electron microscope images of (a) pre-amplification and (b) post-amplification APEX sensors in which exosomes are captured by the sensor via anti-CD63 antibody, and after amplification, soluble substrate (3,3'-diamino). From benzidine tetrahydrochloride), it can be seen that insoluble light deposits are localized and grown. The resulting APEX signal amplification was highly correlated with the increase in area range due to localized deposits. Mass production of APEX microarray sensors. All APEX sensors were manufactured on 8-inch silicon (Si) wafers. The manufacturing process includes: (1) Thermal oxidation to form a 10 nm silicon dioxide (SiO ₂ ) layer and low pressure chemical vapor deposition (LPCVD) to 145 nm silicon nitride (Si ₃ N ₄ ). Was deposited on the wafer. (2) After coating with a photoresist, deep ultraviolet (DUV) lithography was performed to define a nanohole array pattern on the resist. This pattern was transferred to a Si ₃ N ₄ membrane by reactive ion etching (RIE). (3) After removing the photoresist, a thin protective layer (100 nm) of SiO ₂ was deposited on the front side of the wafer by plasma-enhanced chemical vapor deposition (PECVD). A photoresist was spin-coated on the back side of the wafer to allow light transmission; a lithography method was used to define the detection area. (4) After etching Si ₃ N ₄ and SiO ₂ with RIE, Si was etched with potassium hydroxide (KOH) and tetramethylammonium hydroxide (TMAH). (5) After etching, the protected SiO ₂ layer was removed using diluted hydrogen fluoride (DHF) (1: 100). (6) Ti / Au (10 nm / 100 nm) was deposited on the Si ₃ N ₄ membrane. Determining the characteristics of APEX microarray sensors. (A) Photograph of an 8-inch wafer showing the large-scale manufacturing of APEX microarray sensor chips. Each wafer consists of> 2000 detector elements. (B) Scanning electron microscope image of very uniform nanoholes of APEX sensor. The inset is an enlarged view of the nanohole lattice. Gradual spectral changes. The APEX sensor was coupled with either (a) an anti-CD63 antibody for exosome capture or (b) an isotope control antibody. Prior to APEX amplification, all sensors were treated with equal concentrations of exosomes from the neuronal cell line (SH-SY5Y). All sensors showed similar levels of antibody-induced surface functionalization (antibody conjugates), but only the anti-CD63 functionalized sensor demonstrated significant spectral shifts associated with exosome binding and APEX amplification, respectively. rice field. In the control sensor in which no exosome binding was present, APEX amplification induced only a slight spectral change. a.u .: Arbitrary unit. Optimization of APEX sensor performance. (A) Comparison of backside illuminated sensor performance. We compared the SPR transmission intensity, full width at half maximum (FWHM) of spectral peaks, and detection sensitivity of APEX sensors with different nanohole diameters. All sensors were irradiated from the back side to complement APEX enzymatic amplification. The optimal APEX design has a nanohole diameter of 230 nm and a regular periodic pattern of 450 nm in a 100 nm thick gold layer on a silicon nitride film. This two-layer plasmon structure corresponds to SPR excitation by backside irradiation. (B) Changes in the transmission spectrum of the optimized sensor with respect to the increasing index of refraction. Increasing the index of refraction induced a change in the transmission spectrum, shifting the resonance peak to longer wavelengths. (C) Spectral shift showed a linear correlation with the increasing index of refraction. (D) APEX reproducibility and repeatability. APEX enzymatic amplification was performed on the same sample and measured with different users, sensor chips, and measurement times. The measurements showed the following analytical coefficients of variation: intergroup = 2.76%, intragroup = 4.14%, overall = 4.59%. All measurements are performed in 3 or more sets, and in (a) the data are shown as mean ± standard deviation. a.u .: Arbitrary unit, n.s .: No significant difference, Student's t-test. APEX workflow and amplification efficiency. (A) APEX workflow for detecting proteins (extravesicles and intracellular) proteins and miRNAs. (B) APEX amplification efficiency for different molecular targets. APEX signals for the following targets were obtained: extracellular protein, Aβ42 protein; intracellular protein, heat shock protein 90; miRNA, miRNA-9. All signals were normalized to the signal prior to photosubstrate addition to determine the amplification factor. The measurements are carried out in triplets, and in (b) the data are shown as mean ± standard deviation. A fibrous structure in which large Aβ aggregates are collected. After incubating the prepared large Aβ42 aggregates for 2 hours, amyloid fibrils were observed. The formed structure was immunolabeled with gold nanoparticles (15 nm) via an anti-Aβ42 antibody and characterized by transmission electron microscopy. Preparation of BSA control aggregates. (A) Outline of BSA protein aggregation. Small BSA control aggregates and large BSA control aggregates were prepared with varying durations of heating, respectively. (B) Characterization of BSA protein aggregates. The hydrodynamic diameter of the BSA aggregate was determined by dynamic light scattering analysis. Both aggregates showed a single peak size distribution. Small aggregates have a diameter of approximately 15 nm and large aggregates have a diameter of approximately 100 nm. Figure 14: Extracellular vesicles isolated from various cellular sources. (A) Nerve cells (SH-SY5Y), (b) Glial cells (GLI36), (c) Endothelial cells (HUVEC), (d) Monocytes (THP-1), (e) Erythrocytes, (f) Thrombosis, Extracellular vesicles were obtained from (g) prostate-origin epithelial cells (PC-3) and (h) ovary-origin epithelial cells (SK-OV-3), respectively. All vesicles were characterized by nanoparticle tracking analysis. See the description in Figure 14-1. APEX measurements of different circulating Aβ populations. (A) APEX assay configuration to characterize different circulating Aβ populations in clinical plasma samples. The exosome-bound Aβ42 population and the total Aβ42 population were measured in undenatured plasma, and the unbound Aβ42 population was detected in plasma filtrate. (B) Incubation of fibronectin with exosome-bound Aβ42 resulted in only minor changes in APEX signaling. (C) Negative controls (APOE lipoprotein and Aβ42 protein, human serum albumin / HSA) showed negligible signals, demonstrating that the APEX assay is specific for exosome-bound Aβ42. All measurements are performed in triplets and the data are shown as mean ± standard deviation. Tracing of Aβ populations in clinical samples. (A) Exosome-bound Aβ42 population. Aβ42 was concentrated directly from undenatured plasma samples to determine the relative levels of colocalization signals of exosome markers (CD63, CD81, and CD9) and neuronal markers (NCAM, L1CAM, and CHL-1) in captured Aβ42. It was measured. All markers could be detected, but the most highly expressed marker in all clinical samples tested was CD63. (B) Plasma filtrate for characterizing the unbound Aβ42 population. To assess unbound Aβ42 populations, vesicle-free plasma filtrates were prepared using membrane filtration (size cutoff = 50 nm, Nuclepore, Whatman). Determined by nanoparticle tracking analysis, the filtrate showed only a tiny number of vesicles. (C) Plasma filtrate also showed insignificant signals for exosome markers (CD63) and neuronal marker (NCAM), demonstrating the efficient removal of exosomes by filtration. Healthy controls with Alzheimer's disease (AD), mild cognitive impairment (MCI), and non-cognitive impairment (NCI). All measurements are performed in triplets and the data are shown as mean ± standard deviation. Correlation between exosome-binding Aβ42 and localized cerebral amyloid loading. Imaging SUVRs of specific brain regions, namely the band-shaped region in the early stage of AD and the occipital region in the late AD stage, were determined. The APEX measurements of exosome-bound Aβ42 were more consistent with the imaging data in the early AD region (a, R ² = 0.8808) than in the imaging data in the late AD region (b, R ² = 0.6863). Alzheimer's disease (AD, n = 17), mild cognitive impairment (MCI, n = 18), vascular dementia (VaD, n = 9), vascular mild cognitive impairment (VMCI, n = 12), not cognitive impairment Healthy control (NCI, n = 16). All measurements are performed in triplets and the data are shown as mean ± standard deviation. Comparison of PET imaging of clinical subjects with different diagnoses. Patients with different clinical diagnoses (n = 72): AD (n = 17), MCI (n = 18), NCI (n = 16), VaD (n = 9), and VMCI (n = 12) cerebral amyloid plaques PET imaging of the load was performed. The standardized uptake value ratio (SUVR) of global mean plaque deposition could be distinguished between AD clinical groups (AD and MCI) and also from other healthy subjects (NCI) and clinical controls (VaD and VMCI). (** P <0.01, **** P <0.0001, Student's t-test). Extracellular vesicles in clinical samples. (A) Extracellular blood samples from subjects with different clinical diagnoses (AD = 17, MCI = 18, NCI = 16, VaD = 9, VMCI = 12, and acute stroke = 12) as measured by nanoparticle tracking analysis. Typical analysis of vesicles. Comparison of (b) vesicle size and (c) vesicle concentration in clinical blood samples (n = 84). Note that there is no significant difference in vesicle size or concentration between samples with different clinical diagnoses (n.s .: no significant difference, Student's t-test). See description in Figure 19A. See description in Figure 19A. Figure 20: Comparison of APEX assay technology, sensor design, and manufacturing. See the description in Figure 20-1. (A) Inhibits the aggregation of starch-forming proteins. (B) Dynamic light scattering analysis confirmed the monopeak size distribution and size difference when the starch-forming protein was incubated with or without an inhibitor. (C) Real-time sensorgram of exosome binding kinetics. Exosomes have been demonstrated to have a much stronger affinity for larger (untreated) Aβ42 aggregates compared to smaller (inhibitor-treated) Aβ42 aggregates. A real-time sensorgram of exosome binding kinetics is shown. Compared to similarly sized bovine serum albumin (BSA) control aggregates, exosomes are starch-forming proteins (eg, Aβ, APP, α-Syn, IRS-1, tau, APOE, SOD1, TDP-43, Basoon, etc. Fibronectin) More strongly bound to aggregates. Shows the specificity of the APEX assay to measure target miRNA molecules. Assays for miR-9, miR-15b, miR-29b, miR-29c, miR-107, miR-146a, and miR-181c have been developed. Specific detections were demonstrated in all assays. The heat map signal normalized the assay (row).

Detailed Description The present specification discloses a sensor chip and a method for detecting a sample by surface plasmon resonance.

In one aspect, a sensor chip comprising a conductive layer on a membrane support layer is provided in which a plurality of apertures penetrate the conductive layer and the membrane support layer, and the plurality of apertures are the conductive layer and / or the same. It is arranged so that surface plasmon resonance occurs due to irradiation of the membrane support layer.

In one aspect, the design of multi-layered structured materials (conductive metals and supporting substrates) allows for plasmon coupling. This design can accommodate bidirectional excitation of surface plasmon resonance and has comparable SPR performance with bidirectional irradiation (from top and bottom).

As used herein, the term "conductive layer" can be a conductive material that exhibits surface plasmon resonance when excited by electromagnetic energy such as light waves. The conductive material may refer to, for example, a metallic conductive material. The conductive material of such metals may be any metal, such as precious metals, alkali metals, transition metals, and alloys. Examples of conductive materials include, but are not limited to, gold, rhodium, palladium, silver, platinum, osmium, iridium, titanium, aluminum, copper, lithium, sodium, potassium, nickel, metal alloys, indium tin oxide, zinc aluminum oxide. , Zinc gallium oxide, titanium nitride, and graphene. In one aspect, the conductive material is gold, silver, aluminum, sodium, indium, or titanium. The metal may be in its original form or may be coated with an additional layer of protective and reinforcing material.

Conductive materials can be "optically observable" if they exhibit significant scattering intensities in the optical region (ultraviolet to visible to infrared spectra) containing wavelengths from approximately 100 nanometers (nm) to 3000 nm. Conductive materials can be "visually observable" if they are detectable by the human eye, ie show significant scattering intensity in the wavelength band of approximately 380 nm to 750 nm, which is the visible spectrum.

In one aspect, the membrane support layer is a structured membrane support layer.

In one aspect, the membrane support layer is silicon nitride or sodium dioxide. Other supports include substrates that can be patterned to form a coupled multilayer plasmon structure.

Different resonance wavelengths and evanescent wave penetration can be achieved by varying the diameter and periodicity of multiple apertures penetrating the conductive and membrane support layers.

Multiple apertures include symmetrical circular holes, spatially anisotropic shapes, such as ellipses, slits, and any aperture of triangles, rectangles, rectangles, or polygons. A combination of holes of various shapes can also be used. Apertures are less than about 1500 nm, less than about 1400 nm, less than about 1300 nm, less than about 1200 nm, less than about 1100 nm, less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, about 600. Less than nm, less than about 500 nm, less than about 450 nm, less than about 400 nm, less than about 350 nm, less than about 300 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, about 210 Less than nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, about 110 Less than nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or about Can have dimensions or diameters less than 10 nm.

In one aspect, the aperture may have dimensions or diameters from about 150 nm to about 450 nm. In one aspect, the apertures are 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290. nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, Or have dimensions or diameters selected from the group consisting of somewhere in between. In one aspect, the aperture is a hole with a diameter of 230 nm.

The term "periodicity" refers to repeating or repeating at regular intervals by positioning the aperture on the sensor chip. Therefore, the term "periodic" refers to a regular and predetermined pattern of apertures relative to each other.

The surface plasmon resonance sensor chip may include an array of periodic apertures. This regular periodicity can allow tight control of resonant wavelengths and evanescent wave intrusion. In one aspect, the aperture has a periodicity of about 250 nm to about 650 nm. In one aspect, the apertures are 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390. nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, Has periodicity selected from the group consisting of 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, and 650 nm, or anywhere in between. .. In one aspect, the aperture has a periodicity of 450 nm.

In one aspect, the aperture is arranged such that the surface plasmon resonance produced by irradiation has a decay length approximately equal to the diameter of the target of the first recognition molecule.

In one aspect, a conductive layer and a membrane support layer are placed on the substrate, the substrate having voids created in regions adjacent to a plurality of apertures in the substrate in both directions of the conductive layer and / or the membrane support layer. Allows irradiation of, thereby causing surface plasmon resonance.

In one aspect, the sensor chip comprises a first recognition molecule immobilized on the surface of the conductive layer. The first recognition molecule can be immobilized on the surface using techniques well known in the art. For example, the first recognition molecule can be adsorbed on the surface. Alternatively, the surface may be coated with streptavidin or a layer of avidin prior to immobilization of the first recognition molecule. The first recognition molecule may be biotinylated and immobilized on the surface by a streptavidin-biotin bond. In one aspect, the surface can be incubated with polyethylene glycol (PEG) molecules. The surface can be incubated with active (carboxylated) thiol-PEG. The surface can then be activated by carbodiimide cross-linking in the MES buffer mixture in which excess NHS / EDC is dissolved and can bind to the first recognition molecule. In an alternative embodiment, the surface can be incubated with a polyethylene glycol (PEG) mixture containing long-active (carboxylated) thiol-PEG and short-inactivated methylated thiol-PEG. The ratio of long-active (carboxylated) thiol-PEG to short-inactivated methylated thiol-PEG can be optimized for maximum functional binding. The surface can then be activated by carbodiimide cross-linking in the MES buffer mixture in which excess NHS / EDC is dissolved and can bind to the first recognition molecule.

The term "recognition molecule" can refer to a molecule that can specifically bind to a sample. A "recognition molecule" can be an antibody, nucleic acid, peptide, aptamer, small molecule, or other synthetic agent.

The term "specimen" refers to a substance present in a sample that is detected or measured on a sensor chip. "Samples" include cells, viruses, nucleic acids, lipids, proteins, peptides, glycopeptides, nanovesicles, microvesicles, exosomes, extracellular vesicles, sugars, metabolites, or combinations thereof, or tissue states. Can be mentioned. A "specimen" can be, for example, an exosome-bound or associated peptide or nucleic acid biomarker (such as a miRNA). A "specimen" can also be, for example, a cell-protein or protein-nucleic acid complex.

In one aspect, the first recognition molecule is an antibody or fragment thereof. The antibody can be, for example, a pan-exosome marker, or an antibody that recognizes an exosome-associated or bound marker. For example, the antibody can be an exosome-rich and characteristic antibody specific for CD63, CD9, or CD81. Antibodies can also be specific for cell origin specific markers such as CHL1, L1CAM, or NCAM. Antibodies can also recognize biomarkers associated or bound to exosomes. For example, the antibody can be an anti-Aβ antibody that recognizes Aβ, or an antibody that recognizes APP, α-syn, or tau bound or associated with exosomes.

As used herein, the term "antibody" is used, but is not limited to, synthetic antibodies, monoclonal antibodies, recombinant production antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, and the like. Chimeric antibodies, single-stranded Fv (scFv), Fab fragments, F (ab') fragments, disulfide-linked Fv (sdFv) (including bispecific sdFv), and anti-idiotype (anti-Id) antibodies, and any of these. Contains the epitope binding fragment. The antibodies provided herein can be monospecific, bispecific, trispecific, or even more polyspecific. Multispecific antibodies may be specific for different epitopes of a polypeptide, or may be specific for both one polypeptide and one heterologous epitope, eg, a heterologous polypeptide or solid support. good.

The terms "protein" and "polypeptide" are used interchangeably and refer to any polymer (greater than or equal to a dipeptide) of an amino acid linked by a peptide bond or modified peptide bond. Polypeptides with amino acid residues less than about 10-20 are commonly referred to as "peptides". The polypeptides of the invention may contain non-peptide components such as carbohydrate groups. Depending on the cell producing the polypeptide, carbohydrates and other non-peptide substituents can be added to the polypeptide, which depends on the cell type. In the present specification, polypeptides are defined in terms of their amino acid backbone structure, and substituents such as carbohydrate groups are not specifically specified, but they may still be present.

As used herein, a "nucleic acid" can be RNA or DNA, which may be single-stranded or double-stranded, and may be, for example, a nucleic acid, polynucleotide, oligonucleotide, nucleic acid analog encoding the protein of interest. For example, peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA), and the like. Such nucleic acid sequences include, for example, but not limited to proteins that act as transcriptional repressors, antisense molecules, ribosome-encoding nucleic acid sequences, small inhibitory nucleic acid sequences, such as, but not limited to, RNAi. Examples include shRNAi, siRNA, microRNAi (mRNAi), antisense oligonucleotides, and the like.

As used herein, "nanovesicle" can refer to a naturally occurring or synthesized vesicle containing an inner cavity. Nanovesicles may include a lipid bilayer membrane that surrounds the components of the lumen. Nanovesicles include liposomes, exosomes, extracellular vesicles, microvesicles, apoptotic vesicles (or a Blebicles), vacuoles, lysosomes, transport vesicles, secretory vesicles, gas vesicles, matrix vesicles, Alternatively, polyvesicles can be mentioned. Nanometers are less than about 1000 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 450 nm, less than about 400 nm, less than about 350 nm, Less than about 300 nm, less than about 250 nm, less than about 240 nm, less than about 230 nm, less than about 220 nm, less than about 210 nm, less than about 200 nm, less than about 190 nm, less than about 180 nm, less than about 170 nm, Less than about 160 nm, less than about 150 nm, less than about 140 nm, less than about 130 nm, less than about 120 nm, less than about 110 nm, less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 70 nm, It can have dimensions of less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, or less than about 10 nm.

Exosomes are a type of nanovesicle and are also referred to in the art as extracellular vesicles, microvesicles, or microparticles. These vesicles are released from eukaryotic cells to the outside of the cell or separate from the cell membrane to the outside of the cell. The size of these membrane vesicles is non-uniform, with diameters ranging from about 10 nm to about 5000 nm. These small vesicles (approximately 10-l000 nm in diameter, preferably 30-100 nm) released by the exocytosis of intracellular polytopes are referred to in the art as "exosomes". The methods and compositions described herein are equally applicable to other vesicles of any size.

The term "sample" refers to any sample that contains or is tested for the presence of a sample. Such samples include cells, organisms (bacteria, viruses), lysed cells or organisms, cell extracts, nuclear extracts, components of cells or organisms, extracellular fluids, in vitro culture media of cells or organisms, blood, plasma. , Serum, gastrointestinal secretions, urine, ascites, tissue or tumor homogenate, lubricant, stool, saliva, sputum, cyst fluid, sheep's water, cerebrospinal fluid, ascites, alveolar lavage fluid, semen, lymph, tears, pleural effusion, papillae Examples include inhalation fluid, breast milk, skin and airways, intestinal tract, and extracellular sections of the urogenital organs, as well as samples derived from or containing prostate fluid. The sample can be a viral or bacterial sample, a sample taken from an environmental source such as a contaminated water area, an air sample, or a soil sample, as well as a sample from the food industry. The sample may also be a biological sample, which means that the sample is derived from or taken from a living body. The organism may be in vivo (eg, whole organism) or in vitro (eg, cultured and grown cells or organs). "Biological sample" also refers to a cell or cell population from a subject, or an amount of tissue or fluid. The sample is most often removed from the subject, but the term "biological sample" can also refer to cells or tissues that are analyzed in vivo, ie, without being removed from the subject. A "biological sample" often contains cells from a subject, but the term may also refer to a non-cellular portion of a non-cellular biological substance, such as blood, saliva, or urine. Biological samples can be derived from primary, secondary, or metastatic tumor resection, bronchoscopic biopsy, or core needle biopsy, or pleural effusion-derived cell bodies. In addition, fine needle suction biological samples are also useful. In one aspect, the biological sample is a primary ascites cell. Biological samples also include explants derived from patient tissue and primary and / or transformed cell cultures. Biological samples can be prepared by removing the cellular sample from the subject, but were previously isolated (eg, by another person, at another time, and / or for another purpose). ) It can also be done using cells or cell extracts. Storage tissue with a history of treatment or prognosis can also be used. Biological samples include, but are not limited to, tissue biopsy, scraps (eg, scraps in the oral cavity), whole blood, plasma, serum, urine, saliva, cell cultures, or cerebrospinal fluid. Be done. The samples analyzed by the compositions and methods described herein may have been treated for purification or enrichment of the contained exosomes. In one aspect, the sample is blood.

In one aspect, an imaging system is provided that includes a light source, a detector, and a sensor chip as defined herein, the detector is positioned to detect light generated by the light source and transmitted through the sensor chip. ..

In one aspect, a kit containing the sensor chips as defined herein is also provided.

The kit may further include a second recognition molecule specific for the sample captured on the surface of the sensor chip or the sample bound to the captured sample. The kit may contain one or more second recognition molecules that are specific to one or more specimens, respectively, so that one or more specimens can be detected.

The second recognition molecule is (1) signal amplification, (2) co-localization analysis (eg, detecting different targets co-existing in the same vesicle), and (3) specimens based on molecular and histological differences. It may be possible to distinguish between subpopulations.

A second recognition molecule may be attached to the signal amplification moiety, which has the ability to induce the formation of insoluble aggregates with increased optical density compared to the captured sample, and the surface plasmon to be measured. It has the ability to increase the resonance signal. For example, the kit may contain a second recognition molecule that is an antibody (such as an antibody specific for Aβ42). A second recognition molecule may be attached to the horseradish peroxidase enzyme. The kit may further include an enzyme substrate. Horseradish peroxidase enzyme can therefore induce the formation of insoluble aggregates with increased optical density compared to the specimen captured on the surface of the sensor chip.

The term "signal-amplifying molecule" refers to a molecule that can induce the formation of insoluble aggregates on the surface of the sensor chip, thereby increasing the optical density compared to the captured sample. This helps to increase the sensitivity of the sensor chip by causing a larger change in transmission wavelength (spectral shift) or change in transmission intensity when the second recognition molecule binds to the sample on the surface of the sensor chip. .. The "signal amplification molecule" can be, for example, an enzyme such as horseradish peroxidase, which reacts with the enzyme substrate to form insoluble aggregates on the surface of the sensor chip. The "signal amplification molecule" may be a secondary antibody that binds to a second recognition molecule on the surface of the sensor chip to form an aggregate. Secondary antibodies may be further attached to enzymes, gold particles, or macromolecules that help form larger aggregates to increase optical density.

In one aspect, the invention relates to a sensitive analytical platform, the so-called amplified plasmon exosome (APEX), that detects exosome-bound amyloid β (Aβ) directly from a blood sample of a patient with Alzheimer's disease (AD). This method of analysis may utilize transmissive surface plasmon resonance (SPR) and enzymatic conversion of photoproducts in situ to enable complex population analysis. APEX technology may enable profiling of multiparametric in situs of exosome components (eg proteins and miRNAs). The APEX platform is used to measure different circulating Aβ tissue states as well as different circulating Aβ populations (exosome binding, unbound, and whole) and correlate these blood measurements with PET imaging of cerebral amyloid plaque loading. Take.

In one aspect, a method of manufacturing a sensor chip is provided, which method is:
(a) Step of providing the upper membrane support layer;
(b) A step of depositing a conductive layer on the upper membrane support layer;
Including the step of forming a plurality of apertures penetrating the membrane support layer, the plurality of apertures also penetrate the conductive layer, and surface plasmon resonance is caused by irradiation of the conductive layer and / or the upper membrane support layer. Arranged to occur.

The method is
The process of coating the upper surface of the silicon substrate with the upper film support layer and the lower surface with the lower film support layer;
A step of providing a layer of photoresist on the top film support layer; and a step of defining multiple apertures in the photoresist by deep UV lithography (DUV), and a pattern of the plurality of apertures by reactive ion etching (RIE). It may include the step of transferring to the upper membrane support layer.

The method is
A step of removing the photoresist of the upper film support layer, and a step of coating the surface of the upper film support layer with a silicon dioxide protective layer;
A step of coating the lower membrane support layer with a layer of photoresist;
The process of defining the detection area in the photoresist by photolithography;
A step of transferring the pattern of the detection area to the lower membrane support layer by reactive ion etching (RIE);
The process of transferring the pattern of the detection area to the silicon substrate;
A step of removing the protective layer on the surface of the supermembrane support layer with diluted hydrogen fluoride; and a step of depositing the conductive layer on the supermembrane support layer may be further included.

As used herein, the term "detection area" refers to an area of the sensor chip that includes a plurality of holes arranged such that surface plasmon resonance occurs upon irradiation of the plasmon layer and / or the membrane support layer.

As used herein, the term "resist" refers to a thin layer used to transfer an image or pattern onto a substrate on which it is laminated. The resist can be patterned by lithography to form a (sub) micrometer-scale temporary mask that protects the underlying substrate selection area during subsequent processing steps, typically etching. The material used to make this thin layer (typically a viscous solution) is also included in the term resist. Resists are generally mixtures of polymers or precursors thereof specially formulated for a given lithography technique with other small molecules (eg, photoacid generators). The resist used in photolithography is, for example, called a "photoresist". The resist used in electron beam lithography is called "e-beam resist".

In one aspect, a method of detecting a sample in a sample is provided, wherein the method is:
(a) The step of capturing the specimen on the surface of the sensor chip as defined herein; and
(b) Including the step of detecting the binding between the second recognition molecule and the sample captured on the surface of the sensor chip, the second recognition molecule is specific to the sample and compared with the control sample. The increased binding of the second recognition molecule indicates the presence of the sample in the sample.

The method may include (sequential or simultaneous) detection of binding of one or more second recognition molecules specific for one or more specimens. This allows the detection, quantification, and analysis of the tissue state (eg, colocalization) of multiple samples or biomarkers in a sample. Each sample can be recognized by a different set of first and second recognition molecules. The first and second recognition molecules can recognize the same sample or different samples, respectively. Various combinations of the first and second recognition molecules may allow the detection of co-localization of the specimen. This may allow simultaneous detection of multiple specimens and may allow detection of co-localization of these molecules.

In one aspect, the method comprises detecting two or more specimens that are co-localized in the sample. Two or more specimens may be present, for example, intracellularly or intracellularly. Alternatively, two or more specimens may be bound to, for example, the same exosome.

In one aspect, the first recognition molecule is an antibody that recognizes Aβ42 and the second recognition molecule is another antibody that recognizes Aβ42.

In one aspect, the first recognition molecule is an antibody that recognizes Aβ42 and the second recognition molecule is an antibody that recognizes CD63, and co-localization of Aβ42 and CD63 is detected.

The detection of "bonding" between the second recognition molecule and the sample captured on the surface of the sensor chip is obtained by a spectral term shift (change in transmission wavelength) or a change in transmission intensity at a fixed wavelength. For example, a sample captured on the surface of a sensor chip has an initial reference wavelength. After binding of the second recognition molecule, the transmitted wavelength can shift to a longer wavelength.

Changes in the transmission resonance wavelength of the sample (or spectral term shift (Δλ)) or changes in transmission intensity at fixed wavelengths can be compared to the changes observed in the control sample. Thereby, for example, it can be determined whether there is an increased binding between the second recognition molecule and the captured sample.

The "increased binding of the second recognition molecule" in the sample compared to the control sample indicates the change in the spectral shift after the binding of the second recognition molecule, or the change in the transmission intensity at a fixed wavelength, with the sample. It can be determined by comparison with a control sample. Increased spectral shift changes or changes in transmission intensity may indicate that there is an increased binding of the second recognition molecule to the sample.

In one aspect, a spectral shift or an increased change in transmission intensity can refer to a 1.2-fold or greater increase between the subject and the control subject. The term is 1.1x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9 Double, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, 30 times, 31 times, 32 times, 33 times, 34 times, 35 times, 36 times, 37 times, 38 times, 39 times, 40 times, 41 times, 42 times , 43 times, 44 times, 45 times, 46 times, 47 times, 48 times, 49 times, 50 times, 51 times, 52 times, 53 times, 54 times, 55 times, 56 times, 57 times, 58 times, 59 times Double, 60 times, 61 times, 62 times, 63 times, 64 times, 65 times, 66 times, 67 times, 68 times, 69 times, 70 times, 71 times, 72 times, 73 times, 74 times, 75 times, 76 times, 77 times, 78 times, 79 times, 80 times, 81 times, 82 times, 83 times, 84 times, 85 times, 86 times, 87 times, 88 times, 89 times, 90 times, 91 times, 92 times , 93x, 94x, 95x, 96x, 97x, 98x, 99x, and 100x.

The second recognition molecule can be a specimen-specific recognition molecule. A second recognition molecule may be coupled to the signal amplification moiety, which is capable of inducing the formation of insoluble aggregates with increased optical density compared to the captured sample. For example, a sample captured on the surface of a sensor chip has an initial reference wavelength. After binding of the second recognition molecule, the transmitted wavelength can shift to a longer wavelength. When the second recognition molecule binds to the signal amplification moiety, the transmission wavelength can shift to longer wavelengths due to the increase in optical density.

The second recognition molecule may be fused with the signal amplification moiety. Alternatively, the second recognition molecule may be conjugated to the signal amplification moiety.

The signal amplification portion can be an enzyme. In one aspect, the signal amplification moiety is an enzyme. The enzyme can be horseradish peroxidase (HRP), alkaline phosphatase, glucose oxidase, β-lactamase, or β-galactoxidase, or an enzymatic fragment thereof. In one aspect, the enzyme is horseradish peroxidase. In one aspect, the first biorecognition molecule is fused with the signal amplification moiety. For example, the first biorecognition molecule can be an antibody covalently fused to a Western wasabi peroxidase enzyme, which is covalently linked to the antibody by techniques well known in the art.

The method may further include contacting the enzyme with the enzyme substrate. The enzyme substrate can be one capable of forming an insoluble product in the presence of the enzyme or by the action of the enzyme. Horseradish peroxidase (HRP) uses 3-amino-9-ethylcarbazole, 3,3', 5,5'-tetramethylbenzidine, or formulations such as chloronaphthol, 4-chloro-1-naphthol. obtain. These substrates can become insoluble products after the enzymatic reaction of HRP. In one aspect, the enzyme substrate is 3,3'-diaminobenzidine tetrahydrochloride.

In an alternative embodiment, the signal amplification moiety can be a secondary antibody capable of binding to a second recognition molecule. Binding of the secondary antibody to the second recognition molecule can induce the formation of insoluble aggregates.

In one aspect, a first recognition molecule is immobilized on the surface of a surface plasmon resonance sensor chip, which is capable of capturing a sample on the surface of the sensor chip. The specimen can be an exosome bound or associated biomarker. The specimen can be an exosome bound or associated aggregation biomarker. The first recognition molecule can be sample-specific.

In one aspect, the first recognition molecule is an antibody. The antibody can be, for example, a pan-exosome marker, or an antibody that recognizes an exosome-bound or bound marker. For example, the antibody can be an exosome-rich and characteristic antibody specific for CD63, CD9, or CD81. Antibodies can also be specific for cell origin specific markers such as CHL1, L1CAM, or NCAM. Antibodies can also recognize biomarkers associated or bound to exosomes. For example, the antibody can be an anti-Aβ antibody that recognizes Aβ, or an antibody that recognizes APP, α-syn, or tau bound or associated with exosomes.

The term "control sample" refers to a sample that does not contain a sample. A "control sample" can be used to determine if a sample contains the sample of interest as a comparison to the sample.

As used herein, the term "biomarker" is understood to be an agent or element whose presence or level correlates with the event of interest. Biomarkers can be cells, proteins, nucleic acids, peptides, glycopeptides, exosomes, or combinations thereof. For example, a biomarker is an Aβ42 or taupeptide whose presence or level indicates that the subject has or is at risk of developing a neurodegenerative disease or amyloidosis. In another example, a biomarker indicates that its presence or level indicates that the subject has or is at risk of developing a neurodegenerative disease or amyloidosis, bound or associated. ) Aβ42 or taupeptide.

In one aspect, the use of a sensor chip as defined herein for detecting a specimen is provided.

In one aspect, a method of detecting a neurodegenerative disease or amyloidosis of interest is provided, wherein the method is:
(a) The step of bringing the sample into contact with the surface of the sensor chip as defined herein; and
(b) Including the step of detecting the binding between the second recognition molecule and the sample captured on the surface of the sensor chip, the second recognition molecule is specific to the sample and compared with the control target. The increased binding of the second recognition molecule indicates that the subject is suffering from a neurodegenerative disease or amyloidosis.

The term "subject" means any animal, such as any vertebrate or mammal, especially human, and may also be referred to, for example, an individual or a patient.

The term "control subject" refers to a subject who is known not to have neurodegenerative disease or amyloidosis, or who is not at risk of developing neurodegenerative disease or amyloidosis. The "control subject" may be a healthy subject. The "control subject" can be a non-cognitively impaired (NCI) person. The term includes samples taken from control subjects.

In one aspect, the biomarker is an exosome bound or associated biomarker. In one aspect, the biomarker is an exosome binding aggregation biomarker. Biomarkers can be selected from the group consisting of Aβ, APP, α-Syn, or tau, but not limited to. In one aspect, Aβ is Aβ42. In another aspect, Aβ is Aβ40. In one aspect, the biomarker is tau. In some embodiments, the biomarkers are Aβ, APP, α-Syn, CD9, CD63, CD81, ALIX, TSG101, Frotillin-1, Flotillin-2, LAMP-1, HSP70, HSP90, CHL1, IRS-1, It is selected from the group consisting of L1CAM, NCAM, tau, APOE, SOD1, TDP-43, Basoon, fibronectin, DNA, and RNA, or combinations thereof, and complexes to which they are bound.

Neurodegenerative diseases can be selected from the group consisting of Alzheimer's disease and mild cognitive impairment.

The method may further include treating a subject found to be suffering from a neurodegenerative disease or amyloidosis.

The term "treat" as used herein is (1) to prevent or delay the appearance of one or more symptoms of a disease, (2) one or more symptoms of a disease or disease. Inhibiting the development of the disease, (3) alleviating the disease, i.e. resulting in the regression of at least one symptom of the disease or disease, and / or (4) the severity of one or more symptoms of the disease. Can be pointed out to bring about a decline in.

In one aspect, the term "treat" refers to the administration of a drug to slow the progression of a neurodegenerative disease or amyloidosis.

The present specification provides a method for detecting a neurodegenerative disease or amyloidosis of a subject, which comprises the step of detecting the level of an exosome-binding biomarker in a sample taken from the subject and is increased compared to a reference. Levels of exosome-binding biomarkers indicate that the subject is suffering from a neurodegenerative disease or amyloidosis.

In one aspect, a method of detecting a neurodegenerative disease or amyloidosis of a subject is provided, the method comprising detecting the level of an exosome-binding aggregation biomarker in a sample taken from the subject and increasing compared to a reference. Levels of exosome-binding aggregation biomarkers indicate that the subject is suffering from a neurodegenerative disease or amyloidosis.

The method may include detection of one or more exosome-binding biomarkers in the sample. This allows the detection of co-localization or presence of multiple biomarkers present on the same exosome.

Biomarkers can be selected from the group consisting of Aβ, APP, α-Syn, or tau, but not limited to.

The method may include detecting the level of molecular subtypes of exosome-binding biomarkers.

In one aspect, Aβ is Aβ42 or Aβ40.

In one aspect, Aβ is a prefibrillary aggregate. Prefibril Aβ aggregates were found to preferentially bind to exosomes. Increased binding to Aβ. In one aspect, the methods defined herein include the detection of exosomes bound to prefibril Aβ aggregates.

In one aspect, the aggregate biomarker is a prefibrillary aggregate. Aggregation biomarkers can be prefibrillarytic aggregates of Aβ. Alternatively, the aggregation biomarker can be an APP, α-Syn, or tau profibril aggregate.

In one aspect, the criterion is a control subject.

In one aspect, a method of measuring exosome binding as a substitute is provided to determine the prefibrillary tissue state of the protein aggregate, and the increased level of protein aggregate in the prefibrillarytic state compared to the control is the subject. Indicates that the patient has a neurodegenerative disease or amyloidosis.

In one aspect, the method further comprises the step of detecting an exosome biomarker selected from the group consisting of CD63, CD9, and CD81, the exosome biomarker being co-localized with the exosome-binding biomarker. In some embodiments, the exosome biomarkers are Aβ, APP, α-Syn, CD9, CD63, CD81, ALIX, TSG101, fibron-1, fibron-2, LAMP-1, HSP70, HSP90, CHL1, IRS- 1. Selected from the group consisting of L1CAM, NCAM, tau, APOE, SOD1, TDP-43, Basoon, fibronectin, DNA, and RNA, or combinations thereof, and complexes to which they are bound.

In one aspect, the method further comprises the step of detecting a neuronal biomarker selected from the group consisting of NCAM, L1CAM, and CHL-1, the neuronal biomarker co-localizing with an exosome-binding biomarker. There is.

In one aspect, methods are provided for measuring the histological and molecular subpopulations of different biomarkers. For example, the method may include measurement of exosome-binding biomarkers, free (unbound) biomarkers, and total (exosome-bound and unbound) biomarkers. The method may also include measuring the relative concentrations of different biomarkers in order to better predict the disease.

In one aspect, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, mild cognitive impairment, vascular dementia, and mild vascular dementia.

In one aspect, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease and mild cognitive impairment.

The method may further include treating a subject suffering from a neurodegenerative disease or amyloidosis.

The method may also correlate with brain imaging studies such as PET imaging. This includes correlation with imaging of specific brain regions. In one aspect, a method of identifying and measuring a circulating biomarker that correlates with brain imaging (PET) is provided. In one aspect, a method of identifying and measuring a circulating biomarker that correlates with imaging (PET) of a particular brain region is provided.

The present invention can (1) use biomarkers, including individual co-localized markers, for the measurement and characterization of different molecular, biophysical, and histological subpopulations of circulating Aβ. , (2) Circulating exosome-bound Aβ in blood can be strongly correlated with PET imaging (global mean) of Aβ deposition across different patient populations, (3) Circulating exosome-bound Aβ in blood is Aβ across different patient populations. There may be a strong correlation with PET imaging (early AD region, band-shaped region), and (4) by circulating exosome-bound Aβ (Alzheimer's disease, mild cognitive impairment, no cognitive impairment, vascular dementia, vascular mild cognitive impairment, And based on the finding that clinical subgroups (such as acute stroke) can be distinguished.

The method may include administering a therapeutically effective amount of the drug to a subject in need of treatment. The drug can be a cholinesterase inhibitor such as donepezil, Rivastigmate, or galantamine. The drug can also be an NMDA receptor antagonist such as memantine. The drug can be a combination of a cholinesterase inhibitor and an NMDA receptor antagonist, such as a combination of donepezil and memantine. The drug can be a BACE1 inhibitor such as AZD3293, or an antibody such as an anti-amyloid antibody such as aducanumab. The drug can also be an anti-tau drug such as TRx0237 (LMTX). In some embodiments, one or more of the therapeutically effective amounts of the drugs described herein, or a combination of two or more, may be administered to a subject in need of treatment.

In some embodiments, molecules effective in reducing the amount of starch-forming protein aggregates may be potential therapeutic agents. Therefore, in some embodiments, the method is directed to the subject in need of treatment: the following molecules (drugs): methylthioninium chloride, leuco-methylthioniniumbis (hydromethanesulfonate), curcumin, acidofuxin, Epigallocatekin asbestosate, safranal, congored, apigenin, azul C, basic blue 41, (trans, trans) -1-bromo-2,5-bis- (3-hydroxycarbonyl-4-hydroxy) styrylbenzene (BSB) ), Chicago Sky Blue 6B,-Cyclodextrin, Daunomycin Hydrochloride, Dimethyl Yellow, Directed Red 80, 2,2-Dihydroxybenzophenone, Hexadecyltrimethylammonium Bromide (C16), Heminchloride, Hematine, Indometacin, Juglon, Lacmoid, Mecro Cyclone sulfosalicylate, melatonin, mylicetin, 1,2-naphthoquinone, nordihydroguanaletinic acid, R ()-norapomorphin hydrobromid, orange G, o-vanillin (2-hydroxy-3-methoxybenzaldehyde), Fel Phenazine, phthalocyanine, rifamycin SV, phenol red, rolitetracycline, quinacrine mastered dihydrochloride, thioflavin S, ThT, and trimethyl (tetradecyl) ammonium bromide (C17), diallyl tartar, eosin Y, phenofibrate, neocuproin, nystalin. , Octadecylsulfate, and one or more, or a combination of two or more of Rhodamine B may be administered.

Increased levels of exosome-binding biomarkers can refer to a 1.2-fold or greater increase between the subject and the control subject. The term "increased level" is 1.1 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times. , 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22 times, 23 times, 24 times Double, 25 times, 26 times, 27 times, 28 times, 29 times, 30 times, 31 times, 32 times, 33 times, 34 times, 35 times, 36 times, 37 times, 38 times, 39 times, 40 times, 41 times, 42 times, 43 times, 44 times, 45 times, 46 times, 47 times, 48 times, 49 times, 50 times, 51 times, 52 times, 53 times, 54 times, 55 times, 56 times, 57 times , 58 times, 59 times, 60 times, 61 times, 62 times, 63 times, 64 times, 65 times, 66 times, 67 times, 68 times, 69 times, 70 times, 71 times, 72 times, 73 times, 74 times Double, 75 times, 76 times, 77 times, 78 times, 79 times, 80 times, 81 times, 82 times, 83 times, 84 times, 85 times, 86 times, 87 times, 88 times, 89 times, 90 times, It can also refer to an increase selected from the group consisting of 91-fold, 92-fold, 93-fold, 94-fold, 95-fold, 96-fold, 97-fold, 98-fold, 99-fold, and 100-fold.

In one aspect, a method of detecting a subject at risk of developing a neurodegenerative disease is provided, the method comprising detecting the level of an exosome-binding biomarker in a sample taken from the subject and compared to a control subject. Increased levels of exosome-binding biomarkers indicate that the subject is suffering from a neurodegenerative disease.

In one aspect, a method of detecting and treating a neurodegenerative disease or amyloidosis of interest is provided, wherein the method is:
(a) In the step of detecting the level of exosome-binding aggregation biomarker in a sample collected from a subject, the increased level of exosome-binding aggregation biomarker compared to the standard indicates that the subject suffers from neurodegenerative disease or amyloidosis. A process that indicates that you are doing, and
(b) Including the step of treating a subject suffering from a neurodegenerative disease or amyloidosis.

In one aspect, a method of treating a subject's neurodegenerative disease or amyloidosis is provided, wherein the method is:
(a) In the step of detecting the level of exosome-binding aggregation biomarker in a sample collected from a subject, the increased level of exosome-binding aggregation biomarker compared to the standard indicates that the subject suffers from neurodegenerative disease or amyloidosis. A process that indicates that you are doing, and
(b) Including the step of treating a subject suffering from a neurodegenerative disease or amyloidosis.

In one aspect, a method of detecting a neurodegenerative disease or amyloidosis of a subject and slowing its progression is provided.
(a) In the step of detecting the level of exosome-binding aggregation biomarker in a sample collected from a subject, the increased level of exosome-binding aggregation biomarker compared to the standard indicates that the subject suffers from neurodegenerative disease or amyloidosis. A process that indicates that you are doing, and
(b) Including the step of treating a subject suffering from a neurodegenerative disease or amyloidosis.

In one aspect, a method of determining the aggregated state of the biomarker in the sample is provided, the method comprising detecting the level of the exosome-binding biomarker in the sample and increasing the exosome-binding biomarker relative to the reference. The level of the marker is an indicator of the degree of cohesion of the biomarker.

The method may include contacting the sample with the exosome population prior to detecting the level of the exosome-binding biomarker.

In one aspect, the aggregated biomarker in the sample preferentially binds to the exosome as compared to the non-aggregated biomarker.

The sample can be taken from the subject.

In one aspect, the increased biomarker cohesion compared to the reference is an indicator of the subject's neurodegenerative disease or amyloidosis.

The method may further comprise treating a subject suffering from a neurodegenerative disease or amyloidosis.

In one aspect, a method of determining the aggregated state of a biomarker in a sample is provided, the method comprising contacting the sample with an exosome population and detecting the level of the exosome-binding biomarker in the sample. Increased levels of exosome-binding biomarkers relative to the reference are indicators of biomarker aggregation.

Those skilled in the art will appreciate that the invention described herein is capable of modifications and modifications other than those specifically described. It should be understood that the present invention includes all such variants and modifications included in its intent and scope. The invention also includes all the steps, features, compositions, and compounds referred to or shown herein individually or collectively, and any combination of any two or more of such steps or features. ..

Throughout this specification and the claims below, unless otherwise specified in the context, the word "comprise" and its variants such as "comprises" and "comprising" are the integers or steps described, or It should be understood that it means the inclusion of an integer or group of steps, but not the exclusion of any other integer or step, or group of integers or steps.

In the present specification, a reference to any conventional publication (or information obtained from it) or any known matter is such a conventional publication (or information obtained from it) or known matter is referred to herein. It does not acknowledge, admit, or suggest anything that forms part of the general knowledge of the field of endeavor, nor should it be interpreted as such.

Next, specific embodiments of the present invention will be described with reference to the following examples, but such examples are for illustration purposes only and do not limit the scope of the generality described above.

In the present specification, a reference to any conventional publication (or information obtained from it) or any known matter is such a conventional publication (or information obtained from it) or known matter is referred to herein. It does not acknowledge, admit, or suggest anything that constitutes part of the general knowledge of the scope of the attempt to relate to it, nor should it be interpreted as such.

Materials and methods Cell culture. Human cell lines SH-SY5Y (nerve cells), HUVEC (umbilical vein endothelium), THP-1 (monocytes), PC-3 (prostatic epithelium), and SK-OV-3 (ovarian epithelium) from the American Type Culture Collection obtained. GLI36 (glia) and SK-OV-3 were grown in Dulbecco's modified essential medium (DMEM, Gibco). SH-SY5Y was cultured in Dulbecco's modified Eagle's medium: Nutrient Mixture F-12 Medium (DMEM / F12 Gibco). PC-3, THP-1, and HUVEC were grown in F-12K, RPMI-1640, and EGM-2 media, respectively. 10% FBS and penicillin-streptomycin were added to all other media except EGM-2, which was added with 5% fetal bovine serum (FBS).

Isosome isolation and quantification. Cells of passages 1 to 15 were cultured in medium without vesicles (5% depleted FBS) for 48 hours, and then vesicles were collected. All media containing exosomes were filtered through a 0.2 μm membrane filter (Millipore), isolated by fractional centrifugation (first 10,000 g, then 100,000 g) and used for exosome analysis using the APEX platform. To isolate exosomes from blood cells and platelets, blood cells were obtained from the blood fraction and platelets were obtained from platelet-rich plasma. These components were washed with HEPES buffered saline and incubated with 2 mM calcium chloride and 2 μM calcium ionophore (A23187) at 37 ° C. to stimulate exosome production. All vesicles were then collected as described above. A nanoparticle tracking analysis (NTA) system (NS300, Nanosight) was used for independent quantification of exosome concentrations. The exosome concentration was adjusted so that approximately 50 vesicles were in the field of view for optimal measurement. For consistency, all NTA measurements were made with the same system settings.

Manufacture of APEX sensors. The APEX sensor was manufactured on an 8-inch silicon (Si) wafer. Briefly, a 10 nm silicon dioxide (SiO ₂ ) layer was formed by thermal oxidation and 145 nm silicon nitride (Si ₃ N ₄ ) was deposited on the wafer by low pressure chemical vapor deposition (LPCVD). After coating with a photoresist, deep ultraviolet (DUV) lithography was performed to define a nanohole array pattern on the resist. This pattern was transferred to a Si ₃ N ₄ membrane by reactive ion etching (RIE). After removing the photoresist, a thin protective layer of SiO ₂ (100 nm) was deposited on the front side of the wafer by plasma-enhanced chemical vapor deposition (PECVD). A photoresist was spin-coated on the back side of the wafer to allow light transmission; a lithography method was used to define the detection area. Si ₃ N ₄ and SiO ₂ were etched by RIE, and then Si was etched with potassium hydroxide (KOH) and tetramethylammonium hydroxide (TMAH). After etching, the protected SiO ₂ layer was removed using diluted hydrogen fluoride (DHF) (1: 100). Finally, Ti / Au (10 nm / 100 nm) was deposited on the Si ₃ N ₄ membrane. All nanohole dimensions and sensor uniformity were characterized by scanning electron microscopy (JEOL 6701).

Channel assembly. A multi-channel flow cell was manufactured by standard soft lithography. Molds were made using SU-8 negative resist (SU8-2025, Microchem). The photoresist was spin-coated on the Si wafer at 2000 rpm for 30 seconds and baked at 65 ° C and 95 ° C for 2 and 5 minutes, respectively. After exposure to UV light, the resist was baked again and then developed under stirring. The developed mold was chemically treated with trichlorosilane vapor for 15 minutes in a desiccator before continued use. Polydimethylsiloxane polymer (PDMS) and a cross-linking agent were mixed at a ratio of 10: 1 and poured into a SU-8 mold. After curing at 65 ° C. for 4 hours, the PDMS layer was cut out of the mold and assembled to the APEX sensor. All inlets and outlets were made with a 1.1 mm biopsy punch for sample processing.

Optical setup and spectral analysis. A tungsten halogen lamp (Stocker Yale Inc.) was used to irradiate the APEX sensor through a 10X microscope objective lens. The transmitted light was collected by an optical fiber and sent to a spectrometer (Ocean Optics). All measurements were performed at room temperature in a closed box to eliminate ambient light interference. The intensity of the transmitted light was digitally recorded as a count for the wavelength (330 nm to 1600 nm). For spectral analysis, spectral peaks were determined by fitting transmission peaks by local regression using a custom R program. All fits were made locally. In other words, in the case of fitting the point x, the fitting was performed using the points near x weighted by the distance from x. Compared to the multi-order polynominal curve fit, this method was able to eliminate the variability in the results caused by the number of data points analyzed and the data range. To determine the optimum geometry of the sensor (Fig. 10), we used spectral changes that quantify the peak transmission intensity, peak shape (full width at half maximum, FWHM), and detection sensitivity in response to changes in the index of refraction. .. The measured transmission spectra demonstrated uniformity across different sensors, with a standard deviation of baseline spectral peak positions of 0.03 nm. All spectral shifts (Δλ) were determined as changes in transmission spectral peaks and calculated relative to appropriate control experiments (see below for details).

Functionalization of the sensor surface. In order to impart molecular specificity to the APEX sensor, first, the prepared Au surface was subjected to long-active (carboxylated) thiol-PEG and short-inactivated methylated thiol-PEG (Thermo Scientific) (activity: inert 1: 1. 3. Incubated for 2 hours at room temperature with a polyethylene glycol (PEG) mixture containing 10 mM in PBS). After washing, the surface was activated by carbodiimide cross-linking in a MES buffer mixture in which excess NHS / EDC was dissolved and bound to specific probes and ligands (eg, antibodies and Aβ42 aggregates). All probe information is listed in Table 1. Excess unbound probe was removed by PBS washing. The combined sensor was stored at 4 ° C in PBS for later use. All sensor surface modifications were observed spectrally to confirm that they were uniformly functionalized.

(Table 1) List of markers used for profiling and their probes

APEX signal amplification. To establish APEX amplification, we incorporated enzymatic growth of insoluble photoproducts for signal enhancement and optimized the photosubstrate concentration and reaction duration to build the platform. Briefly, exosomes were incubated with CD63 functionalized APEX sensors (BD Biosciences) for 10 minutes. The bound vesicles were then labeled with biotinylated anti-CD63 antibody (Ancell, 10 minutes). As a control experiment, the amplification efficiency was determined using the same amount of biotinylated IgG isotope control antibody (Biolegend) for the bound vesicles. After washing the unbound antibody, the sensitive horseradish peroxidase bound to Neutravidin (Thermo Scientific) is reacted with the bound vesicles and then as a photosubstrate in different concentrations of 3,3'-diaminobenzidine tetrahydrochloride ( Life Technologies) was introduced. Real-time spectral changes were observed to determine optimal substrate concentration and reaction duration. The optimization condition was determined to be 1 mg / mL for 3 minutes. Incubation and wash flow rates were all maintained at 3 μl / min and 10 μl / min, respectively. Localized adhesion of insoluble photoproducts was confirmed by scanning electron microscopy. This optimization workflow is shown in Figure 11a.

Using this set of conditions, known amounts of exosomes were further titrated and their associated APEX signals were measured. We determined that the APEX detection limit as the lowest target concentration that could generate a detection signal = 3 x (standard deviation of the background signal from the control).

APEX protein detection. All sensor surfaces were blocked with 2% w / v bovine serum albumin (BSA) to reduce non-specific protein binding. Exosomes were placed in a functionalized sensor and incubated at room temperature for 10 minutes to capture exosomes and washed with PBS to remove unbound components. For extracellular protein targets, exosomes were directly labeled with the detection antibody for APEX amplification, as described above. For intravesicular protein targets, exosomes were subjected to additional immobilization and permeabilization (eBioscience) before labeling with the detection antibody. Spectral measurements were performed before and after APEX amplification and analyzed with a specially designed R program.

APEX miRNA detection. The APEX sensor was functionalized with the p19 protein (New England Biolabs) by its chitin-binding domain and blocked at 2% w / v BSA. For miRNA detection, exosome lysates were incubated with a biotinylated RNA probe (350 nM) for 15 minutes to hybridize to the target miRNA strand. This mixture was fed into a functionalized sensor in a binding buffer (1 x p19 Binding Buffer, pH 7.0, 40U RNase inhibitor, 0.1 mg / mL BSA) to capture p19 of hybridized miRNA target / RNA probe duplexes. Made possible. For APEX amplification, the bound biotinylated duplex was charged with highly sensitive horseradish peroxidase bound to Neutroavidin (Thermo Scientific). Spectral measurements were performed and analyzed with a specially designed R program.

Enzyme-linked immunosorbent assay (ELISA). The captured antibody (5 μg / ml) was adsorbed on an ELISA plate (Thermo Scientific), blocked with Superblock (Thermo Scientific), and then incubated with the sample. After washing with PBST (PBS containing 0.05% Tween 20), the detection antibody (2 μg / ml) was added and incubated at room temperature for 2 hours. After incubation with a secondary antibody (Thermo Scientific) bound to horseradish peroxidase and a chemiluminescent substrate (Thermo Scientific), the chemiluminescence intensity was measured to detect the protein (Tecan).

Western blotting. Exosomes isolated by ultracentrifugation were dissolved in a radioactive immunoprecipitation assay (RIPA) buffer containing a protease inhibitor (Thermo Scientific) and quantified by the bicinchoninic acid assay (BCA assay, Thermo Scientific). Protein lysates are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membrane (PVDF, Invitrogen), and protein markers: HSP90 (Cell Signaling), HSP70 (BioLegend), Flotilin 1 Immunoblots were performed using antibodies against (BD Biosciences), CD63 (Santa Cruz), ALIX (Cell Signaling), TSG101 (BD Biosciences), LAMP-1 (R & D Systems), and the nerve cell marker NCAM (R & D Systems). After incubation with a secondary antibody (Cell Signaling) bound to horseradish peroxidase, immunodetection was performed by enhanced chemiluminescence (Thermo Scientific).

Protein aggregation. The lyophilized NH ₄ OH treated Aβ42 protein (rPeptide) was resuspended in NaOH (60 mM, 4 ° C.) and sonicated to adjust the pH in PBS34 to pH 7.4. The protein was immediately filtered through a 0.2 μm membrane filter (Millipore) and the filtrate was used as smaller Aβ42 aggregates. For the preparation of larger Aβ42 aggregates, the proteins were treated as described above and incubated for 1 hour with stirring to induce further aggregation and then filtered through a 0.2 μm membrane filter (Millipore). The filtrate was used as a large Aβ aggregate. To prepare BSA aggregates of similar size as controls, 2% w / v BSA was dissolved in PBS and heated at 80 ° C. for 1 hour and 2 hours, respectively, with smaller and larger control aggregates. Induced clustering.

Agglomerate sizing. The hydrodynamic diameters of Aβ42 and BSA aggregates were determined by dynamic light scattering analysis (Zetasizer Nano ZSP, Malvern). Measurements were performed 3 x 14 times at 4 ° C. Z mean diameter and polydispersity were analyzed. The autocorrelation function and polydispersity were observed for each measurement to ensure the quality of the sizing sample.

を用いて、エクソソームと相互作用する結合凝集体の総表面積を計算した。式中、Sはシグナル、zはセンサー表面からの距離、Eはz = 0のときの電場であり、この場合の定数ldは減衰長さであって、現センサー設計では200 nmに設定され、また、rは結合したタンパク質凝集体の半径である。 Characterization of exosome-Aβ binding. The prepared protein aggregates (Aβ42 and BSA controls) were used for surface functionalization of the APEX sensor via EDC / NHS binding as described above. Unbound protein aggregates were rinsed with PBS. From the obtained transmission spectrum shift, the amount of protein complex was measured. This information was used to determine the number of bound protein aggregates and the total bounded protein surface area for their exosome binding (see below for more information) to normalize the binding affinity. Following surface functionalization with protein aggregates, exosomes (10 ¹⁰ / ml) were charged into the sensor. Spectral changes were measured every 3 seconds for a total of 480 seconds to construct a real-time dynamic sensorgram. The exosome binding kinetics and binding affinity of protein aggregates of different sizes were determined. To illustrate the difference in protein aggregate size, as well as the exponential decay of SPR-related sensitivity (with increasing distance from the detection surface):

Was used to calculate the total surface area of binding aggregates that interact with exosomes. In the equation, S is the signal, z is the distance from the sensor surface, E is the electric field when z = 0, and the constant ld in this case is the decay length, set to 200 nm in the current sensor design. Also, r is the radius of the bound protein aggregate.

All protein aggregates were close to spheres (Fig. 3b, left), as can be seen from transmission electron micrographs, and their r was determined by dynamic light scattering analysis. The above formula was used to determine the number of protein aggregates bound to the sensor, as well as the total surface area of each, thereby estimating the number of binding sites available for interaction with exosomes. All exosome binding data (Δλ) were normalized for each protein binding site. Normalized Aβ42 binding data were relativized for BSA controls of similar size and fitted to determine the binding affinity constant KD.

Scanning electron microscopy. All samples were fixed with half-strength Karnovsky fixative and washed twice with PBS. After dehydration with a series of elevated ethanol, the sample was transferred, subjected to supercritical drying (Leica), spatter coated with gold (Leica), and then imaged with a scanning electron microscope (JEOL 6701).

Transmission electron microscopy. Exosomes were immunolabeled with gold nanoparticles (15 nm, Ted Pella), fixed with 2% paraformaldehyde and transferred to a copper grid (Ted Pella). The bound vesicles were washed and counterstained with a mixture of uranyl oxalate and methylcellulose. The dried sample was imaged with a transmission electron microscope (JEOL 2200FS).

Collection of clinical samples. This study was approved by the NUH and NUS Clinical Trial Review Boards (2015/00441, 2015/00406, and 2016/01201). All subjects were recruited and given informed consent according to a protocol approved by the Review Board. All recruited subjects underwent several neuropsychological assessments at the National University Hospital (NUH, Singapore), including the Mini-Mental State Examination (MMSE), Montreal Cognitive Assessment (MoCa), and vascular dementia to assess cognitive performance. A dementia battery (VDB) was included. The clinical diagnosis of AD, MCI, or NCI was made by neuropsychological assessment, as well as evaluation of clinical features and blood studies. The clinical diagnosis of VaD and VMCI was made from a combination of neuropsychological assessments, a clinical history of stroke, and the degree of cerebrovascular disease observed by magnetic resonance imaging (MRI). All clinical assessments and classifications were performed according to publication criteria 46-48, independent of APEX measurements. Plasma samples of acute stroke were collected from patients diagnosed with stroke within 24 hours of admission. Long-term plasma samples were collected from patients during a one-year follow-up outpatient visit and were not imaged by PET. For plasma collection, subject's venous blood (5 ml) was collected in an EDTA tube and treated immediately (if applicable) prior to injection of the PET radiotracer. Briefly, all blood samples were centrifuged at 400 g (4 ° C) for 10 minutes. Plasma was transferred without destroying the buffy coat and centrifuge again at 1100 g (4 ° C) for 10 minutes. All plasma samples were unspecified and stored at -80 ° C until measurements on the APEX platform. All APEX measurements were performed with blinded PET imaging results and clinical diagnosis.

Clinical APEX measurement. All plasma samples were measured according to the assay configuration outlined in Supplement Figure 15a. Briefly, undenatured plasma samples were used directly by Aβ42 capture and CD63 detection (Aβ42 + CD63 +) without the need for vesicle purification or isolation to measure exosome-bound Aβ42 populations. Large slags were removed by size exclusion filtration (cutoff size = 50 nm, Whatman) to indicate the presence of unbound Aβ42 populations in plasma samples. This was necessary because unbound Aβ42 and total Aβ42 could not be distinguished in this assay configuration based on Aβ42 capture and Aβ42 detection. Plasma filtrates were evaluated by Aβ42 capture and Aβ42 detection (Aβ42 + Aβ42 +) to measure unbound Aβ. Note that this filtration was performed only to demonstrate the presence of the unbound Aβ42 population and is not necessary in the clinical context of directly measuring only the more reflective exosome-bound Aβ42 from undenatured plasma samples. To measure total Aβ42, undenatured plasma samples were directly evaluated by Aβ42 capture and Aβ42 detection (Aβ42 + Aβ42 +). In all measurements, 5% BSA was used as the blocking agent for the APEX sensor. Also included were sample-compatible negative controls incubating the same sample on a control sensor functionalized with an IgG isotype control antibody. All measurements were relativized with respect to this IgG control to reveal sample-aware non-specific binding.

Imaging by positron emission tomography (PET). After blood collection, the subjects were scanned using the Siemens 3T Biograph mMR system (Siemens Healthineers), and PET images and MR images were acquired at the same time. PET data were obtained 40-70 minutes after intravenous injection of 370 MBq of 11C-Pittsburgh Compound B (PiB). MR data was acquired using a 12-channel head receive coil for acquisition, with Ultrashort Echo Time (UTE) images for PET attenuation correction and T1-weighted magnetization-prepared gradient echo (MPRAGE) images (1 mm isotropic resolution). , TI / TE / TR = 900 / 3.05 / 1950 ms).

Analysis of PET data. T1-weighted MPRAGE images were processed with Freesurfer (5.3.0) to obtain cortical segmentation for PET data analysis. PET images were restored using the Ordinary Poisson Ordered Subset Expectation Maximization (OP-OSEM) algorithm and smoothed using a 4 mm Gaussian filter. Data were attenuated using a μ-map based on UTE. Next, the obtained standardized uptake value (SUV) image after attenuation correction was co-aligned with the MPRAGE image using Advanced Normalization Tools (ANTs), and the average cerebellar gray material intensity was used using the subject-specific Freesurfer region segmentation. The standardized uptake value ratio (SUVR) was calculated relative to the relative. The global mean SUVR of each patient was calculated by calculating the mean SUVR of a specific region and averaging the SUVR of the whole brain region.

Statistical analysis. All measurements are performed in triplets and the data are shown as mean ± standard deviation. A significance test was performed by Student's two-sided t-test. In the sample-to-sample comparison, multiple pairs of samples were tested, and the obtained P-value was adjusted for multiple hypothesis testing by Bonferroni correction. A value having adjusted P <0.05 was determined to be significant. Analytical and biological coefficients of variation (ie, intragroup, intergroup, and overall) were determined by a paired one-way ANOVA test. In this clinical study, correlation was performed with linear regression to determine goodness of fit (R2). All statistical analyzes were performed using the R-package (version 3.4.2) and Graphpad Prism 7.

Example 1
Amplified plasmon analysis of exosome-bound Aβ
One of the earliest pathological features of AD is Aβ deposition in the brain. These plaques are formed from clustering of aberrant amyloid protein fragments, primarily the hydrophobic splicing variant Aβ42. Aβ protein is released into the extracellular space and can enter the bloodstream and circulate. Exosomes, also found in the extracellular space, are nanoscale membrane vesicles secreted by mammalian cells through fusion of polyvesicular endosomes with the cell membrane. During this exosome renewal, glycoproteins and glycolipids are incorporated into the invaginated cell membrane and are divided into new exosomes 10 and 11. These surface markers allow exosomes to associate and bind to extracellular Aβ proteins (Fig. 1a). Exosome morphology, size distribution, and molecular composition of extracellular vesicles derived from neuronal origin (SH-SY5Y cells) were confirmed by multimodal characterization (Fig. 5). Permeational electron microscopy of vesicles further revealed their ability to bind to Aβ42 protein aggregates (Fig. 1b and Fig. 5).

To evaluate exosome-Aβ binding, we have developed an APEX platform for amplified multiparametric profiling of exosome molecule co-localization. The system measures transmissive SPR with a periodic array of plasmon nanoholes patterned as a two-layer photonic structure and rapidly grows insoluble photoproducts on bound exosomes by enzymatic conversion of in situ. (Fig. 1c). To complement APEX enzymatic adhesion (occurring on the top of the sensor), pattern plasmon nanoholes of the same size on the bound bilayer photonic system and enhance SPR measurements with backside irradiation (away from enzymatic activity) (Figure 20). did. The resulting enzymatic adhesion not only stably changes the index of refraction for SPR signal amplification, but also molecular co-stationary, as demonstrated by the redshift of the transmitted light spectrum (spectral shift Δλ, FIG. 1d). Spatially defined with respect to presence analysis. Scanning electron microscopy images of exosomes bound to the sensor before and after APEX amplification confirmed the localized growth of photoadhesions after enzymatic conversion (Fig. 6).

Using the APEX platform developed in this way, the binding of Aβ protein to exosomes was measured directly from clinical blood samples of AD patients and control subjects, and the measured values were correlated with PET imaging of global and localized brain plaque deposits. Taken (Fig. 1e). For high-throughput complex clinical analysis, advanced manufacturing methods (ie, deep UV lithography, Figure 7) are used to fabricate sensor microarrays on 8-inch wafers, with each wafer having more than 40 microarrays with> 2000 detector elements. The chip could be accommodated (Fig. 8a). Figure 1f shows a photograph of the developed APEX microarray chip used for parallel measurements in this study. From the scanning electron microscope image of the developed sensor, it was found that the production was very uniform (Fig. 8b).

Optimized signal amplification for complex profiling First, we developed enzymatic APEX amplification. A series of sensor functionalizations, namely antibody binding, exosome binding, enzyme labeling, and photoproduct amplification, were performed and a stepwise total spectral shift (cumulative Δλ, FIG. 2a) was measured. The sensor was functionalized with an antibody against CD63, a type III lysosomal membrane protein rich and characteristic of exosomes, to capture vesicles derived from nerve cells (SHSY5Y). To promote localized adhesion of insoluble photoproducts, horseradish peroxidase was incorporated as a cascade enzyme and catalyzed the conversion of its soluble substrate (3,3'-diaminobenzidine tetrahydrochloride). The vesicles bound to the sensor were enzymatically labeled with another anti-CD63 antibody. Enzyme labeling did not result in significant spectral changes, but photoproduct formation resulted in approximately 400% signal enhancement. In comparison, control experiments with IgG isotope control antibodies demonstrated subtle background changes (Fig. 9). Importantly, this SPR signal amplification was highly correlated with the increase in area range due to highly localized light deposits when confirmed by scanning electron microscopy (Fig. 2b).

To complement the enzymatic amplification (occurs on the top of the sensor), the APEX sensor design has been optimized to improve its analytical performance and stability. The two-layer plasmon structure of APEX allows SPR excitation by backside illumination (Fig. 2c), as compared to the ready-made gold-supported glass design for surface irradiation only (Fig. 20). This new optimized design not only showed strong transmissive SPR with backside irradiation (Figs. 10a-c), but also analytical stability, probably due to the reduction of irradiation directly incident on the enzyme activity (ie, temperature fluctuations). Demonstrated (Fig. 10d). The APEX assay was further established by optimizing the enzyme substrate concentration and reaction duration (Fig. 2d). With constant backside illumination, real-time spectral changes associated with different substrate concentrations were observed, and it was found that significant signal amplification was achieved in less than 10 minutes and thus the entire APEX workflow could be completed in less than an hour. Was done.

These optimization conditions were met, and then the APEX detection sensitivity for exosome quantification was measured. Neuron (SH-SY5Y) -derived vesicles were quantified by standard nanoparticle tracking analysis. A titration experiment was performed using an anti-CD63 antibody (Fig. 2e). Optimized APEX amplification was determined to be able to increase and enhance detection sensitivity by 10-fold, establishing a detection limit (LOD) of approximately 200 exosomes. This observed sensitivity is the highest LOD in bulk exosome measurements reported so far, and is 105 ^- fold and 103 ^- fold better than Western blotting and chemiluminescent ELISA, respectively.

We have further developed an assay to profile various markers associated with neurodegenerative diseases using the microarray APEX platform. Amyloid β (Aβ42), amyloid precursor protein (APP), alpha-synuclein (α-syn), L1 closely related homologue (CHL1), insulin receptor substrate 1 (IRS-1), nerve cell adhesion molecule (NCAM), and A specific assay for protein markers such as tau protein (Fig. 2f) was established. Importantly, with the development of further APEX assay workflows (Figure 11a), this platform demonstrated the ability to amplify signals for the detection of both extracellular and intracellular proteins as well as exosome miRNAs (Figure 11b). All detection probes used in assay development are listed in Table 1.

Increased binding of Aβ aggregates to exosomes Next, the developed APEX platform was used to evaluate the binding of exosomes to pathological Aβ proteins of different structural forms. To mimic the various stages of amyloid dissemination and fibrosis, aggregates of different sizes of Aβ42, the key component of amyloid plaque, were prepared. Different sizes of Aβ42 aggregates were formed with varying degrees of clustering (Fig. 3a, see Methods for experimental details), and their spherical morphology and monopeak size distribution were measured by transmission electron microscopy and transmission electron microscopy, respectively. Confirmed by dynamic light scattering analysis (Fig. 3b). It was further noted that the larger Aβ42 aggregates demonstrated a strong tendency to form fibrous structures (Fig. 12).

To determine the kinetics of exosome-Aβ binding, the prepared Aβ42 aggregates were immobilized on the APEX platform and the sensor was incubated with an equal concentration of neuronal cell-derived exosomes (Fig. 3c). For comparison, in a control experiment, bovine serum albumin (BSA) aggregates of similar size (Fig. 13) were prepared and characterized. By measuring exosome binding in real time, it was demonstrated that exosomes can bind to Aβ42 aggregates more strongly than similarly sized BSA controls, regardless of aggregate size (Fig. 3d). More importantly, vesicles have significantly higher (more than 5-fold) affinities for larger Aβ42 aggregates compared to their binding affinity for smaller Aβ42 aggregates (Figure 3d, left). Shown (Fig. 3d, right). All affinities were normalized to the surface area of the Aβ42 aggregate and relativized to each BSA control (see Methods for details).

Extracellular vesicles from various cell origins were then used and the binding of each to the larger Aβ42 aggregate was measured using the APEX platform (Fig. 3e). Equal concentrations of vesicles from various cell origins (Fig. 14) determined by nanoparticle tracking analysis were incubated with the Aβ42 functionalized sensor. Of all cell origins tested, vesicles from nerve cells, erythrocytes, platelets, and epithelial cells demonstrated stronger binding to Aβ42 aggregates, while vesicles from glial and endothelial cells were insignificant. It was noted that it showed only binding. A set of specific markers for each of these cellular origins, as well as pan-exosome markers (ie, CD63), were then used to amplify APEX signals in bound vesicles. CD63 was consistent in performance across all vesicles tested for signal enhancement. With this marker identification, we developed an APEX assay using CD63 to identify and measure exosome-binding Aβ (defined as Aβ42 + CD63 +; Figure 15).

Brain plaque loading revealed by blood exosome-binding Aβ Exosome-binding Aβ is a circulating biomarker that more closely reflects brain plaque loading in view of the increased binding of exosomes to prefibril Aβ aggregates, which are components of amyloid plaque. I made the hypothesis that it could be. To test this hypothesis, different APEX assays were developed using different antibodies to evaluate different circulating Aβ42 populations from clinical blood samples (Fig. 15a). Specifically, to characterize the exosome-bound Aβ42 population, the APEX assay was designed to concentrate Aβ42 directly from undenatured plasma and measure the relative amount of CD63 bound to captured Aβ42. This assay configuration not only demonstrated specific detection of the Aβ42 + CD63 + population (Figs. 15b-c), but also reflected functional aptitude, with increased binding of prefibril Aβ aggregates to exosomes to bind CD63. The signal could be regarded as a surrogate indicator for measuring the relative amount of prefibrils Aβ42 in the total circulation Aβ42. To indicate the presence of an unbound Aβ42 population, large size filters (eg, exosomes) in plasma were removed by size removal filtration before measuring Aβ42 in plasma filtrate. Finally, undenatured plasma was evaluated directly by Aβ42 enrichment and Aβ42 detection to measure total circulation Aβ42.

Next, (1) can APEX measure circulating Aβ42 directly from blood samples, (2) how different blood Aβ42 populations correlate with brain plaque load, and (3) specific circulating Aβ42 populations vary clinically. Possibility clinical trials were conducted with the aim of addressing the main question of whether the groups could be distinguished.

To achieve these goals, AD has been diagnosed (n = 17), mild cognitive impairment has been diagnosed (MCI, n = 18), non-cognitive impairment healthy controls (NCI, n = 16), and vascular dementia. Clinical controls of disease (VaD, n = 9) and neurovascular dementia (ie, vascular mild cognitive impairment, VMCI, n = 12; and acute stroke, n = 12), subjects of the same age (n = 84) Recruited. All clinical information is given in Table 2. All subjects were recruited for blood sampling and APEX analysis. With the exception of patients with acute stroke, all subjects also agreed to perform PET imaging of cerebral amyloid plaque at the same time. Plasma samples were collected shortly before injection with a Pittsburgh Compound B (PiB) radiotracer for PET imaging. PET imaging revealed a wide range of brain plaque loads between and within the imaging clinical groups (Figure 4a), demonstrating local brain diversity (Table 2) and consistent with other published clinical studies. ..

AD:アルツハイマー病、MCI:軽度認知障害、NCI:認知障害なし、VaD:血管性認知症、VMCI:血管性軽度認知障害 (Table 2) Standardized uptake value ratio (SUVR) for clinical information and PET imaging

AD: Alzheimer's disease, MCI: Mild cognitive impairment, NCI: No cognitive impairment, VaD: Vascular dementia, VMCI: Vascular mild cognitive impairment

The developed APEX assay (Figure 11a) was used to evaluate different circulating Aβ42 populations in these clinical plasma samples: exosome-bound Aβ42, unbound population, and total circulation Aβ42 (Figure 4b). The exosome-bound Aβ42 population exhibits strong co-localization signals by exosome markers (ie, CD63, CD9, and CD81) and neuronal markers (ie, NCAM, L1CAM, and CHL-1), in which neuronal exosomes It was suggested that it could occupy a large proportion (Fig. 16a). Since measurements of unbound Aβ42 were performed on plasma filtrates, these filtrates were further characterized to confirm the number of their tiny vesicles and the microcolocalization of signals by exosome and neuronal markers. (Figs. 16b-c). Correlated with global PET amyloid imaging, exosome-bound Aβ42 measurements were compared to unbound Aβ42 (Fig. 4b, center, R ² = 0.0193) or total Aβ42 (Fig. 4b, right, R ² = 0.1471). The highest correlation was shown (Fig. 4b, left, R ² = 0.9002). Interestingly, unlike the low and negative associations (shown in this study and other published reports) demonstrated by all Aβ42 measurements, relative CD63 from the exosome-bound Aβ42 population. The measurements showed a high correlation and positive association with PET imaging of cerebral amyloid plaques. This finding showed that (1) exosomes showed increased binding to prefibril Aβ42 aggregates, especially larger aggregates that could easily form fibrils (Fig. 3d), and (2) PET tracers showed larger amyloid sources. It is believed to be due to the Aβ42 binding priority of similar exosomes and PET tracers that it binds well to fibers but rarely binds to smaller aggregates. This high correlation also demonstrates brain region specificity, and the measured value of exosome-bound Aβ42 is that of the brain in the band-shaped region (early AD region, Fig. 17a) rather than the occipital region (late AD region, Fig. 17b). It showed a stronger correlation with plaque load.

Regarding the distinction between clinical diagnoses, only APEX analysis of exosome-bound Aβ42, not the unbound or total Aβ42 population, demonstrated good specificity (Fig. 4d). Specifically, measurements of exosome-bound Aβ42 distinguish between AD clinical groups (ie AD and MCI, P <0.01) as well as other healthy and clinical controls (P <0.0001, Student's t-test). did it. This demonstrated specificity is comparable to PET brain amyloid imaging in terms of distinction between different clinical groups (Fig. 18). On the other hand, nanoparticle tracking analysis of extracellular vesicles of plasma showed no significant difference in vesicle size or concentration across all clinical groups (Fig. 19).

Example 2
AD is the most common form of severe dementia. Due to its complex and progressive neuropathology, early detection and timely intervention are essential for the success of disease-altering treatments. Although there is a great deal of interest in finding serum biomarkers for AD, their development is confused due to several challenges. First, unlike its equivalent in cerebrospinal fluid, pathological AD molecules in the bloodstream have been demonstrated to be much lower in concentration. Plasma Aβ levels tend to be close to the lower limit of the detection limit of conventional ELISA assays, and this limitation may have led to some discrepancies in the findings in the published reports. Second, little correlation has been established between plasma Aβ analysis and brain plaque deposition, which is the earliest pathological feature of AD. One of the possible reasons may be due to different measurement methodologies. PET imaging probes, commonly used to determine cerebral amyloid loading, preferentially measure the attachment of insoluble fibrils, whereas conventional ELISA measurements measure soluble Aβ in plasma. In addition, the potential correlation between blood-based measurements and brain pathology may have been obscured by previous uniform blood measurements. However, this difference depends on the more fundamental question: whether there is a subpopulation of circulating Aβ protein that can better reflect the pathology of fibrils in the brain.

To distinguish between different circulating Aβ populations, we have developed a dedicated analytical platform (APEX) for multiparametric analysis of exosome-bound Aβ, unbound Aβ, and total Aβ directly from plasma. Specifically, new technological advances in sensor design, device manufacturing, and assay development will be used to achieve increased optical performance and detection (Figure 20). In terms of sensor design and manufacturing, the APEX platform features a periodic array of gold nanoholes on a patterned silicon nitride film and is manufactured by deep UV lithography, the latest manufacturing process for large-scale precision nanopatterning. To. Thanks to these technological advances, APEX technology has (1) improved optical performance (ie, increased transmission intensity that enables SPR detection with two-way light irradiation), and (2) reliable large-scale production. To realize. In terms of assay technology, the APEX platform leverages rapid in situ enzymatic conversion to achieve highly localized amplification signals. This development not only enables sensitive detection of diverse targets (eg intravesicular proteins and RNA targets), but also because insoluble deposits are locally formed only when multiple targets are present in the exosome at the same time. , Facilitates exosome colocalization analysis in multiparametric population studies. As a result of these technological advances, the observed sensitivity of APEX is the highest ever reported for exosome profiling, far surpassing standard ELISA measurements. Using the developed APEX platform, we demonstrated increased binding of exosomes to the larger profibrillar Aβ, a major component of fibrillar amyloid plaques. Further identification and quantification of the exosome-binding amyloid subpopulation (CD63 + Aβ42 +) in clinical plasma samples reveals a variety of clinical populations (ie, AD, MCI, cognitively normal controls, as well as other neurodegenerative diseases and nerves). High correlation with cerebral amyloid plaque load was found across clinical controls with vascular disease).

Assessing different circulating Aβ populations can lead to a paradigm shift in AD research and clinical care. Accumulated evidence supports that prefibrillar Aβ aggregates can function as toxic drivers for AD neurodegeneration. Recent findings on their preferential binding to exosomes, as well as the enrichment of exosome markers in human amyloid plaques, not only shed light on possible new plaque seeding mechanisms, but also more reflect complex AD pathology. It also suggests the importance of exosome-binding Aβ as a circulating biomarker. Therefore, this study may complement other preclinical and clinical studies in terms of technological development and improvement of biomarkers. In terms of technological development, for example, IP mass spectrometry enables unbiased molecular screening and detection of different molecular isoforms and variants (eg (APP) 669-711 and Aβ1-40), especially for the discovery of biomarkers. However, this APEX technique is targeted because it provides fast and sensitive readings from unmodified plasma samples and does not require the extensive sample processing typically required for mass spectrometric measurements. Suitable for clinical measurement. In terms of biomarker improvements, analysis of different circulating Aβ populations, as demonstrated in this study, can reveal novel correlations previously hidden by uniform blood measurements and are based on future blood. Can advance AD clinical management. Importantly, this methodology developed could be enhanced in more than 400 AD trials currently underway to redefine the current standard of care for patients. The developed technology enables objective evaluation of disease modification treatment in different phases of clinical trials through additional technological innovations such as on-chip exosome treatment, combination analysis with other AD markers, and long-term clinical cohort validation. All together, it could provide a comprehensive ability to facilitate early detection, molecular stratification, and continuous observation of significant minimal invasiveness.

Example 3
This example shows that incubation of starch-forming protein aggregates with an inhibitor reduced spontaneous protein aggregation.

Method Agglomerate sizing. The hydrodynamic diameters of starch-forming protein aggregates and BSA aggregates were determined by dynamic light scattering analysis (Zetasizer Nano ZSP, Malvern). Measurements were performed 3 x 14 times at 4 ° C. Z mean diameter and polydispersity were analyzed. The autocorrelation function and polydispersity were observed for each measurement to ensure the quality of the sizing sample.

Protein aggregation. The lyophilized starch-forming protein was resuspended in NaOH (60 mM, 4 ° C.) and sonicated to adjust the pH to pH 7.4 in PBS. The protein was immediately filtered through a 0.2 μm membrane filter (Millipore) and the filtrate was used as small initial aggregates. For the preparation of larger aggregates, the proteins were treated as described above and incubated for 1 hour with stirring to induce further aggregation. For inhibitor treatment, 10 μM inhibitor (eg, methylene blue) was added to the protein before incubation. After incubation was complete, aggregate sizing was performed.

Optical analysis. For experimental analysis, the APEX sensor was back-illuminated through a 10X microscope objective with a tungsten halogen lamp (Stocker Yale Inc.). The transmitted light was collected by an optical fiber and sent to a spectrometer (Ocean Optics). All measurements were performed at room temperature in a closed box to eliminate ambient light interference. The intensity of the transmitted light was digitally recorded as a count against the wavelength. For spectral analysis, spectral peaks were determined by fitting transmission peaks by local regression using a custom R program. All fits were made locally. In other words, in the case of fitting the point x, the fitting was performed using the points near x weighted by the distance from x. Compared to the multi-order polynominal curve fit, this method was able to eliminate the variability in the results caused by the number of data points analyzed and the data range. All spectral shifts (Δλ) were determined as changes in transmission spectral peaks and calculated relative to appropriate control experiments.

Exosome-protein binding characterization. The prepared protein aggregates (Aβ42 and BSA controls) were used for surface functionalization of the APEX sensor via EDC / NHS binding as described above. Unbound protein aggregates were rinsed with PBS. From the obtained transmission spectrum shift, the amount of bound protein was measured. This information was used to determine the number of bound protein aggregates and the total bounded protein surface area for their exosome binding (see below for more information) to normalize the binding affinity. Following surface functionalization with protein aggregates, exosomes were introduced into the sensor. Spectral changes were measured every 3 seconds for a total of 480 seconds to construct a real-time dynamic sensorgram. The exosome binding kinetics and binding affinity of protein aggregates of different sizes were determined.

を用いて、エクソソームと相互作用する結合凝集体の総表面積を計算した。式中、Sはシグナル、zはセンサー表面からの距離、Eはz = 0のときの電場であり、この場合の定数ldは減衰長さであって、現センサー設計では200 nmに設定され、また、rは結合したタンパク質凝集体の半径である。 To illustrate the difference in protein aggregate size, as well as the exponential decay of SPR-related sensitivity (with increasing distance from the sensor surface):

Was used to calculate the total surface area of binding aggregates that interact with exosomes. In the equation, S is the signal, z is the distance from the sensor surface, E is the electric field when z = 0, and the constant ld in this case is the decay length, set to 200 nm in the current sensor design. Also, r is the radius of the bound protein aggregate.

All protein aggregates were close to spheres and their r was determined by dynamic light scattering analysis. The above formula was used to determine the number of protein aggregates bound to the sensor, as well as the total surface area of each, thereby estimating the number of binding sites available for interaction with exosomes. All exosome binding data (Δλ) were normalized for each protein binding site. Normalized Aβ42 binding data were relativized for BSA controls of similar size and fitted to determine the binding affinity constant _KD .

miRNA analysis. Exosome lysates were incubated with a biotinylated RNA probe followed by RNA duplex capture and APEX signal amplification on a p19 functionalized APEX sensor.

Results The pathology of several neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis involves aggregation of misfolded starch-forming proteins. Treatments that target protein aggregation include various strategies to clear aggregated starch-forming proteins, such as disintegrating starch-forming protein aggregates or inhibiting starch-forming protein aggregation. Methylthioninium chloride and leuco-methylthioninium bis (hydromethane) have been shown to be effective in reducing the amount of starch-forming protein aggregates and are therefore potential potential therapeutic agents for disease modification. Sulfonate), curcumin, eosin, eosinate epigalocatecin, safranal, congo red, apigenin, azul C, basic blue 41, (trans, trans) -1-bromo-2,5-bis- (3-hydroxycarbonyl) -4-Hydroxy) Styrylbenzene (BSB), Chicago Skyblue 6B, -Cyclodextrin, Daunomycin Hydrochloride, Dimethyl Yellow, Directred 80, 2,2-Dihydroxybenzophenone, Hexadecyltrimethylammonium Bromide (C16), Heminchloride, Hematin , Indomethasin, Juglon, Lacmoid, Mecrocycline sulfosalicylate, Melatonin, Mylicetin, 1,2-naphthoquinone, Nordihydroguayaletinic acid, R ()-Noruapomorphin hydrobromid, Orange G, o-vanillin (2- Hydroxy-3-methoxybenzaldehyde), ferfenazine, phthalocyanine, rifamycin SV, phenol red, rolitetracycline, quinacrine mustard dihydrochloride, thioflavin S, ThT, and trimethyl (tetradecyl) ammonium bromide (C17), diallyl tartar, eosin Y, Examples include phenofibrate, neocuproin, nystalin, octadecylsulfate, and rhodamine B.

Since no animal model has been demonstrated showing accurate pathology that reflects AD pathology in the human brain, in vitro experiments were used to model the effect of disease-modifying treatments on inhibiting starch-forming protein aggregation. The initial size of the starch-forming protein was confirmed by dynamic light scattering analysis, and then the aliquot of the protein was incubated with or without the inhibitor. In the absence of inhibitors, the size of the protein aggregates increased with increasing incubation duration as a result of spontaneous aggregation of starch-forming proteins. However, in the presence of inhibitors, the increase in size as a result of spontaneous aggregation of proteins was small, resulting in the formation of smaller protein aggregates.

After incubation, the starch-forming protein was functionalized on the surface of the sensor chip and then incubated with neuronal exosomes. As can be seen from the difference in binding affinity, neuronal exosomes were observed to bind preferentially to larger protein aggregates, demonstrating less binding to smaller protein aggregates treated with inhibitors. .. Since exosome-protein binding can be a surrogate indicator of the biophysical and / or biochemical properties of proteins affected by disease-modifying therapies, the APEX platform provides an effective assessment of disease-modifying therapies. enable.

Although the configurations of some examples have been described, various modifications, alternative configurations, and equivalents may be used without departing from the spirit of the present disclosure. For example, the elements described above may be components of a larger system, in which case other rules may take precedence over the application of the present invention or other modifications may be made. In addition, some steps may be performed before, during, or after the above-mentioned factors are examined.

All publications, sequence accession numbers, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

Exemplary Aspects of the Disclosure In some aspects and aspects, the following are described herein.

A sensor chip comprising a conductive layer on a membrane support layer, wherein a plurality of apertures penetrate the conductive layer and the membrane support layer, and the plurality of apertures are the conductive layer and / or the film support layer. It is arranged so that surface plasmon resonance occurs due to irradiation.

In some embodiments, the conductive layer is gold, silver, aluminum, sodium, indium, or titanium.

In some embodiments, the membrane support layer is silicon nitride or sodium dioxide.

In some embodiments, the aperture has a diameter of about 150 nm to about 450 nm.

In some embodiments, the apertures are arranged periodically.

In some embodiments, the aperture has a periodicity of about 250 nm to about 650 nm.

In some embodiments, the first recognition molecule is immobilized on the surface of the conductive layer.

In some embodiments, the aperture is arranged such that the surface plasmon resonance produced by irradiation has a decay length approximately equal to the diameter of the target of the first recognition molecule.

In some embodiments, the conductive layer and the membrane support layer are placed on the substrate, the substrate having voids created in the regions adjacent to the plurality of apertures in the substrate, the conductive layer and / or the membrane support layer. Allows irradiation in both directions, thereby causing surface plasmon resonance.

In another aspect, the present specification describes an imaging system comprising a light source, a detector, and a sensor chip according to any one of 1-9, wherein the light source is arranged to illuminate the sensor chip. The detector is positioned to detect the light transmitted through the sensor chip.

In another aspect, the present specification describes a method of manufacturing a sensor chip, wherein the method is described.
(a) Step of providing the upper membrane support layer;
(b) A step of depositing a conductive layer on the upper membrane support layer;
The main step of forming a plurality of apertures penetrating the film support layer is included, and the plurality of apertures also penetrate the conductive layer, and by irradiation of the conductive layer and / or the upper film support layer. It is arranged so that surface plasmon resonance occurs.

In some embodiments, the method is
The process of coating the upper surface of the silicon substrate with the upper film support layer and the lower surface with the lower film support layer;
A step of providing a layer of photoresist on the top film support layer; a step of defining multiple apertures in the photoresist by deep UV lithography (DUV), and a step of defining the pattern of the plurality of apertures by reactive ion etching (RIE). The step of transferring to the upper membrane support layer is included.

In some embodiments, the method is
A step of removing the photoresist of the upper film support layer, and a step of coating the surface of the upper film support layer with a silicon dioxide protective layer;
A step of coating the lower membrane support layer with a layer of photoresist;
The process of defining the detection area in the photoresist by photolithography;
A step of transferring the pattern of the detection area to the lower membrane support layer by reactive ion etching (RIE);
The process of transferring the pattern of the detection area to the silicon substrate;
It further comprises a step of removing the protective layer on the surface of the supermembrane support layer with diluted hydrogen fluoride; and a step of depositing the conductive layer on the supermembrane support layer.

In another aspect, the present specification describes a method of detecting a sample in a sample, wherein the method is described.
(a) A step of capturing a sample on the surface of the sensor chip according to any one of 1 to 9; and
(b) Including the step of detecting the binding between the second recognition molecule and the sample captured on the surface of the sensor chip, the second recognition molecule is specific to the sample and compared with the control sample. The increased binding of the second recognition molecule indicates the presence of the sample in the sample.

In some embodiments, the second recognition molecule is sample-specific, the second recognition molecule is bound to a signal amplification moiety, which is compared to the optical density of the captured specimen. It has the ability to induce the formation of insoluble aggregates with increased optical density.

In some embodiments, the second recognition molecule is fused with the signal amplification moiety.

In some embodiments, the signal amplification moiety is an enzyme.

In some embodiments, the enzyme is horseradish peroxidase.

In some embodiments, the method further comprises contacting the enzyme with an enzyme substrate.

In some embodiments, the signal amplification moiety is a secondary antibody capable of binding to a second recognition molecule.

In some embodiments, the first recognition molecule is immobilized on the surface of a surface plasmon resonance sensor chip, which is capable of capturing the specimen on the surface of the sensor chip.

In some embodiments, the specimen is an exosome-binding biomarker.

In some embodiments, the method comprises detecting two or more specimens that are co-localized in the sample.

In another aspect, the specification describes a kit comprising the sensor chips described herein.

In some embodiments, the kit comprises a second recognition molecule that detects the specimen captured on the surface of the sensor chip.

In some embodiments, the second recognition molecule is attached to a signal amplification moiety that induces the formation of insoluble aggregates with increased optical density compared to the optical density of the captured specimen. Have the ability.

In another aspect, the present specification describes a method of detecting a neurodegenerative disease or amyloidosis of interest, which method is described.
(a) The step of bringing the sample derived from the subject into contact with the surface of the sensor chip according to any one of 1 to 9; and
(b) Including the step of detecting the binding between the second recognition molecule and the sample captured on the surface of the sensor chip, the second recognition molecule is specific to the sample and can be used as a control sample or a control target. The increased binding of the second recognition molecule in comparison indicates that the subject suffers from a neurodegenerative disease.

In some embodiments of the method, the second recognition molecule is attached to a signal amplification moiety, which forms an insoluble aggregate with an increased optical density compared to the optical density of the captured specimen. Has the ability to guide.

In some embodiments, the specimen is an exosome-binding biomarker.

In some embodiments, the specimen is an exosome-binding biomarker, or a biomarker contained within an exosome.

In some embodiments, the biomarkers are Aβ, APP, α-Syn, CD9, CD63, CD81, ALIX, TSG101, Frotillin-1, Flotillin-2, LAMP-1, HSP70, HSP90, CHL1, IRS-1, It is selected from the group consisting of L1CAM, NCAM, tau, APOE, SOD1, TDP-43, Basoon, fibronectin, DNA, and RNA, or combinations thereof, and complexes to which they are bound.

In some embodiments, Aβ is Aβ42, Aβ40, Aβ39, or Aβ38.

In some embodiments, the neurodegenerative disease is Alzheimer's disease, mild cognitive impairment, dementia, vascular dementia, Parkinson's disease, muscular atrophic lateral sclerosis, multiple sclerosis, progressive supranuclear palsy, tauopathy. , And / or selected from the group consisting of mild vascular dementia.

In some embodiments, the method further comprises treating a subject found to be suffering from a neurodegenerative disease.

In some embodiments, the therapeutic step comprises administering to the subject a therapeutically effective amount of one or more drugs, or a combination thereof.

In some embodiments, the drug is a cholinesterase inhibitor such as donepezil, rivastigmate, or galantamine; an NMDA receptor antagonist such as memantine; a combination of a cholinesterase inhibitor and an NMDA receptor antagonist, such as a combination of donepezil and memantine; BACE1 inhibitors such as AZD3293; antibodies such as aducanumab; or antitau drugs such as TRx0237 (LMTX).

In some embodiments, the drug is methylthioninium chloride, leuco-methylthioninium bis (hydromethanesulfonate), curcumin, acid fuxin, epigallocatekin asbestosate, safranal, congo red, apigenin, azul C, basic. Blue 41, (Trans, Trans) -1-bromo-2,5-bis- (3-Hydroxycarbonyl-4-hydroxy) Stylylbenzene (BSB), Chicago Sky Blue 6B, -Cyclodextrin, Daunomycin Hydrochloride, Dimethyl Yellow , Directred 80, 2,2-Dihydroxybenzophenone, Hexadecyltrimethylammonium bromide (C16), Heminchloride, Hematine, Indomethacin, Juglon, Lacmoid, Mecrocycline sulfosalicylate, Melatonin, Mylicetin, 1,2-Naftquinone, Nor Dihydroguayaletinic acid, R ()-norapomorphin hydrobromid, orange G, o-vanillin (2-hydroxy-3-methoxybenzaldehyde), ferfenazine, phthalocyanine, rifamycin SV, phenol red, rolitetracycline, quinacrine mustard dihydro It is selected from chloride, thioflavin S, ThT, and trimethyl (tetradecyl) ammonium bromide (C17), diallyl tartar, eosin Y, phenofibrate, neocuproin, nystalin, octadecylsulfate, and / or Rhodamine B.

Claims (34)

A sensor chip comprising a conductive layer on a membrane support layer, wherein a plurality of apertures penetrate the conductive layer and the membrane support layer, and the plurality of apertures are the conductive layer and / or the film support layer. A sensor chip that is arranged so that surface plasmon resonance occurs when irradiated. The sensor chip according to claim 1, wherein the conductive layer is gold, silver, aluminum, sodium, indium, or titanium. The sensor chip according to claim 1 or 2, wherein the film support layer is silicon nitride or sodium dioxide. The sensor chip according to claim 1, wherein the aperture has a diameter of about 150 nm to about 450 nm. The sensor chip according to any one of claims 1 to 4, wherein the aperture is periodically arranged. The sensor chip according to claim 5, wherein the aperture has a periodicity of about 250 nm to about 650 nm. The sensor chip according to any one of claims 1 to 6, comprising a first recognition molecule immobilized on the surface of the conductive layer. The sensor chip according to claim 7, wherein the aperture is arranged such that the surface plasmon resonance generated by irradiation has an attenuation length substantially equal to the diameter of the target of the first recognition molecule. The conductive layer and the film support layer are arranged on the substrate, and the substrate has voids formed in a region adjacent to the plurality of apertures on the substrate, and the conductive layer and / or the film support layer has a gap. The sensor chip according to any one of claims 1 to 8, which enables irradiation in both directions and thereby causes surface plasmon resonance. An imaging system comprising a light source, a detector, and the sensor chip according to any one of claims 1 to 9, wherein the light source is arranged to illuminate the sensor chip, and the detector is: An imaging system positioned to detect light transmitted through the sensor chip. A method of manufacturing sensor chips, the following main steps:
(a) Step of providing the upper membrane support layer;
(b) A step of depositing a conductive layer on the upper membrane support layer;
(c) Including the step of forming a plurality of apertures penetrating the membrane support layer.
A method in which the plurality of apertures also penetrate the conductive layer and are arranged such that surface plasmon resonance occurs upon irradiation of the conductive layer and / or the upper membrane support layer. A step of coating the upper surface of the silicon substrate with the upper film support layer and the lower surface of the lower film support layer;
A step of providing a layer of photoresist on the top film support layer; a step of defining a plurality of apertures on the photoresist by deep UV lithography (DUV), and a step of defining the pattern of the plurality of apertures by reactive ion etching (RIE). 11. The method of claim 11, comprising the step of transferring to the upper membrane support layer. A step of removing the photoresist of the upper film support layer, and a step of coating the surface of the upper film support layer with a silicon dioxide protective layer;
A step of coating the lower membrane support layer with a layer of photoresist;
The process of defining the detection area on the photoresist by photolithography;
A step of transferring the pattern of the detection area to the lower membrane support layer by reactive ion etching (RIE);
The process of transferring the pattern of the detection area to the silicon substrate;
12. The method according to claim 12, further comprising a step of removing the protective layer on the surface of the upper film support layer with diluted hydrogen fluoride; and a step of depositing the conductive layer on the upper film support layer. It is a method of detecting a sample in a sample.
(a) The step of capturing the sample on the surface of the sensor chip according to any one of claims 1 to 9; and
(b) Including the step of detecting the binding between the second recognition molecule and the sample captured on the surface of the sensor chip.
A method, wherein the second recognition molecule is specific to the sample and the increased binding of the second recognition molecule as compared to the control sample indicates the presence of the sample in the sample. The second recognition molecule is specific to the sample, the second recognition molecule is bound to the signal amplification portion, and the signal amplification portion has an optical density as compared with the optical density of the captured sample. 14. The method of claim 14, wherein is capable of inducing the formation of increased insoluble aggregates. The method of claim 14 or 15, wherein the second recognition molecule is fused with the signal amplification moiety. 15. The method of claim 15 or 16, wherein the signal amplification moiety is an enzyme. 17. The method of claim 17, wherein the enzyme is horseradish peroxidase. 17. The method of claim 17 or 18, further comprising contacting the enzyme with an enzyme substrate. 15. The method of claim 15 or 16, wherein the signal amplification moiety is a secondary antibody capable of binding to the second recognition molecule. Claims 15-20, wherein the first recognition molecule is immobilized on the surface of the surface plasmon resonance sensor chip, and the first recognition molecule is capable of capturing the sample on the surface of the sensor chip. The method described in any one of the above. 21. The method of claim 21, wherein the specimen is an exosome-binding biomarker. 14. The method of claim 14, comprising the step of detecting two or more samples co-localized in the sample. A kit comprising the sensor chip according to any one of claims 1 to 9. 24. The kit of claim 24, comprising a second recognition molecule for detecting a sample captured on the surface of the sensor chip. The second recognition molecule is bound to a signal amplification moiety, which is capable of inducing the formation of insoluble aggregates with an increased optical density relative to the optical density of the captured specimen. Item 25 The kit. A method for detecting a neurodegenerative disease or amyloidosis in a subject.
(a) The step of bringing the sample collected from the subject into contact with the surface of the sensor chip according to any one of claims 1 to 9; and
(b) Including the step of detecting the binding between the second recognition molecule and the sample captured on the surface of the sensor chip.
The second recognition molecule is specific to the sample, and the increased binding of the second recognition molecule relative to the control sample or control subject indicates that the subject suffers from a neurodegenerative disease. Method. The second recognition molecule is bound to the signal amplification moiety, which is capable of inducing the formation of insoluble aggregates with an increased optical density relative to the optical density of the captured specimen. 27. The method of claim 27. 28. The method of claim 27 or 28, wherein the specimen is an exosome-binding biomarker. 28. The method of claim 27 or 28, wherein the sample is an exosome-binding biomarker, or a biomarker contained within an exosome. The biomarkers are Aβ, APP, α-Syn, CD9, CD63, CD81, ALIX, TSG101, Flotillin-1, Flotillin-2, LAMP-1, HSP70, HSP90, CHL1, IRS-1, L1CAM, NCAM, Tau. , APOE, SOD1, TDP-43, Basoon, fibronectin, DNA, and RNA, or combinations thereof, and a complex to which they are bound, according to any one of claims 27-30. Method. 31. The method of claim 31, wherein the Aβ is Aβ42, Aβ40, Aβ39, or Aβ38. The neurodegenerative diseases include Alzheimer's disease, mild cognitive impairment, vascular dementia, dementia, Parkinson's disease, muscular atrophic lateral sclerosis, multiple sclerosis, progressive supranuclear palsy, tauopathy, and mild vascular dementia. The method according to any one of claims 27 to 31, selected from the group consisting of cognitive impairment. 28-32. The method of claim 28-32, further comprising treating a subject found to be suffering from a neurodegenerative disease.

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